Chimeric immunogenic polypeptides

ABSTRACT

Provided herein are chimeric polypeptides that may be used, e.g., for the diagnosis of or vaccination against  Ehrlichia chaffeensis  and/or  Ehrlichia canis.

This application claims the benefit of U.S. Provisional PatentApplication No. 62/711,005, filed Jul. 27, 2018, the entirety of whichis incorporated herein by reference.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the field of molecularbiology and medicine. More particularly, it concerns chimericpolypeptides that may be used for diagnostic or vaccination purposes.

2. Description of Related Art

Human monocytotropic ehrlichiosis (HME) is a group 1 NIAID emergingdisease, and the etiologic agent, E. chaffeensis, is classified as aCategory C priority pathogen. HME is an undifferentiated febrile illnessthat is life-threatening, clinical diagnosis is difficult, anddefinitive diagnosis is most often retrospective (Walker and Dumler,1997; Walker et al., 2004; Dumler et al., 2007). Although well over8,000 cases have been reported to the Centers for Disease Control as of2012, this number likely underestimates the actual number of cases by100-fold (Olano et al., 2003). The disease is often undiagnosed due tothe non-specific symptoms associated with the onset, but it results inpatient hospitalization in 43-62% of cases (Fishbein et al., 1994).Progression of the disease can result in a fatal outcome and ofteninvolves multisystem failure, with acute respiratory distress syndrome(ARDS) and meningoencephalitis being common in many fatal cases(Fishbein et al., 1994; Paparone et al., 1995). The threat to publichealth is increasing with newly emerging ehrlichial agents, yet vaccinesfor human ehrlichioses are not available, and therapeutic options arelimited. New information and bioinformatics prediction tools have beendeveloped that make a genome-wide identification of protectiveimmunodiagnostic/vaccine candidates feasible (He et al., 2010; Magnan etal., 2010)

Prospects for development of effective subunit vaccines andimmunodiagnostics for Ehrlichia have been limited due to many factors,not the least of which is the small repertoire ofimmunoreactive/protective proteins that have been molecularly defined(McBride and Walker, 2010). The gaps in knowledge required to addressthis problem for Ehrlichia chaffeensis have been narrowed by progress inunderstanding of protective/pathologic immune mechanisms (Feng andWalker 2004; Nandi et al., 2007; Winslow et al., 2000), immunomolecularcharacterization of some vaccine/diagnostic antigens (Kuriakose et al.,2012; Li et al., 2002), genome, transcriptome and proteome profiles(Kuriakose et al., 2011; Lin et al., 2011), new animal models (Winslowet al., 1998; Sotomay et al., 2001), and other technological advances.Studies utilizing low throughput approaches to define antigeniccomponents of E. chaffeensis have yielded a small group of protectiveantigens that include a major outer membrane protein (OMP), and a familyof secreted tandem repeat protein (TRP) effectors with major protectivelinear antibody epitopes (Kuriakose et al., 2012; Li et al., 2001).Nevertheless, these antigens likely represent a significant, butincomplete repertoire of immunoreactive/protective proteins. Inaddition, it is well established that antibody-mediated immunity isnecessary for protection against E. chaffeensis infection (Winslow etal., 2000; Li et al., 2002; Kuriakose et al., 2012; Li et al., 2001;Racine et al., 2011; Yager et al., 2005), and antibodies are thecornerstone of the most effective vaccines for humans. Elimination of E.chaffeensis occurs, at least in part, during the extracellular stage ofinfection (Li and Winslow 2003); however, intracellular immunemechanisms may also be important, and defining the characteristics ofantigens/antibodies that are protective in both environments is criticalfor effective vaccine development. Clearly, there is a need for new andimproved methods for diagnosing and vaccinating against E. chaffeensisand E. canis.

SUMMARY OF THE INVENTION

The present disclosure, in some aspects, provides methods andcompositions for the diagnosis of and vaccination against Ehrlichiachaffeensis (E. chaffeensis) and/or Ehrlichia canis (E. canis). In someembodiments, chimeric immunogenic peptides and polypeptides areprovided. In some aspects, it has been discovered that contiguousrepetition of different immunogenic sequences can be used to generatepeptides or polypeptides that display improved properties for diagnosisand/or inducing immune responses against E. chaffeensis and/or E. canis.

As shown in the below examples, immunoreactive E. chaffeensis and E.canis peptides were used to produce chimeric Ehrlichia polypeptides.Chimeric polypeptides included in Table 2 were verified to beimmunoreactive using ELISA tests on human monocytotropic ehrlichiosis(HME) positive human and canine sera. ELISA testing using positive HMEsera obtained from patients revealed that the below constructs elicitedsignificant responses, indicating that peptides (e.g., of Table 1) canbe used to produce chimeric polypeptides (e.g., of Formula I or in Table2) that may be used in diagnostic methods to detect infection by E.chaffeensis or E. canis, or may be used to induce an immune response ina subject (e.g., a human or a dog) against E. chaffeensis or E. canis.

An aspect of the present invention relates to an isolated polypeptide,wherein the isolated polypeptide comprises: (i) at least two of theimmunogenic sequences of Table 1, or a sequence at least 90% identical(preferably at least 95% identical); and (ii) wherein at least one ofthe immunogenic sequences is contiguously repeated in the polypeptide.For example, in some embodiments, at least 2, 3, 4, 5, 6, or 7immunogenic sequences are contiguously repeated in the isolatedpolypeptide. In some embodiments, each of the at least two immunogenicsequences of Table 1, or a sequence at least 90% identical arecontiguously repeated 1, 2, 3, 4, 5, 6, or 7 times in the polypeptide.In some embodiments, the isolated polypeptide comprises one or more of(SEQ ID NOs:11-16 or 36-42), wherein the one or more of (SEQ IDNOs:11-16 or 36-42) are contiguously repeated 0, 1, 2, or 3 times. Insome embodiments, each of the immunogenic sequences are contiguouslyrepeated from 1 to 3 times in the polypeptide. In some embodiments, eachof the immunogenic sequences are contiguously repeated from 1 to 2 timesin the polypeptide. In some embodiments, the isolated polypeptidecomprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all of thefollowing immunogenic sequences: TRP120 (SEQ ID NO:22), TRP140 (SEQ IDNO:23), A34N1 (SEQ ID NO:7), TRP63 (SEQ ID NO:18), TRP47 (SEQ ID NO:17),TRP75 (SEQ ID NO:19), TRP28 (SEQ ID NO:2), TRP36R1 (SEQ ID NO:3),TRP36R2 (SEQ ID NO:4), TRP36R3 (SEQ ID NO:6), TRP36CO (SEQ ID NO:36),TRP19 (SEQ ID NO:1), HSP (SEQ ID NO:24), or a sequence at least 90%identical (preferably at least 95% identical); wherein each of theimmunogenic sequences are contiguously repeated from 1 to 7 times in thepolypeptide. The isolated polypeptide may comprise TRP36R1 and TRP140.In some embodiments, the TRP36R1 is contiguously repeated 4-8 times, andwherein the TRP140 is contiguously repeated 1-3 times. The polypeptidemay comprise or consists of 8 repeats of TRP36R1 and 4 repeats ofTRP140. The polypeptide may comprise or consist of SEQ ID NO:27. Thepolypeptide may further comprise TRP19. The polypeptide may comprise orconsist of SEQ ID NO:26. In some embodiments, the isolated polypeptidecomprises at least two, at least three, at least four, at least five orall of the immunogenic sequences: TRP32, TRP120, TRP36R1, TRP140, TRP28,and/or HSP. In some embodiments, the TRP36R1 if present is repeated 2-6times, and wherein the other immunogenic sequences are repeated 1-3times. In some embodiments, the polypeptide comprises all of TRP32,TRP120, TRP36 (such as TRP36R1, TRP36R2, TRP36R3, and/or TRP36CO),TRP140, TRP28, and HSP. The polypeptide may comprise or consist of SEQID NO:28. In some embodiments, the polypeptide comprises TRP120, TRP36,TRP140, and TRP28. The polypeptide may comprise or consists of SEQ IDNO:29. In some embodiments, the isolated polypeptide comprises at leastthree, at least four, at least five or all of TRP32R1, TRP32R2, TRP32R3,TRP32R4, TRP120, and A34N1. In some embodiments, TRP120 and A34N1 areeach contiguously repeated 1, 2, or 3 times. The polypeptide maycomprise or consist of SEQ ID NO:25. The polypeptide may comprise atleast two, at least three, or all of A34N1, TRP63, TRP47, and/or TRP75.The polypeptide may comprise A34N1 and TRP63. Each of the immunogenicsequences may be contiguously repeated 1-2 times. The polypeptide maycomprise A34N1, TRP63, and TRP75. The polypeptide may comprise orconsist of SEQ ID NO:34. In some embodiments, the polypeptide comprisesA34N1, TRP63, and TRP47. The polypeptide may comprise or consist of SEQID NO:33. In some embodiments, the polypeptide comprises at least two,at least three, or all of A34N1, TRP63, TRP47, and/or TRP75. Thepolypeptide may comprise A34N1 and TRP63. In some embodiments, each ofthe immunogenic sequences are contiguously repeated 1-2 times. Thepolypeptide may comprise A34N1, TRP63, and TRP75. The polypeptide maycomprise or consist of SEQ ID NO:34. The polypeptide may comprise A34N1,TRP63, and TRP47. The polypeptide may comprise or consist of SEQ IDNO:33. In some embodiments, the polypeptide comprises at least two, atleast three, or all of TRP120, A34N1, TRP47, and/or TRP63. Thepolypeptide may comprise A34N1 and TRP120. Each of the immunogenicsequences may be contiguously repeated 1-2 times. The polypeptide maycomprise A34N1, TRP120, and TRP63. The polypeptide may comprise orconsist of SEQ ID NO:31. The polypeptide may comprise A34N1, TRP120, andTRP47. The polypeptide may comprise or consist of SEQ ID NO:32. Thepolypeptide may comprise A34N1, TRP120, TRP47, and TRP63. Thepolypeptide may comprise or consist of SEQ ID NO:30. In someembodiments, the polypeptide comprises at least two, at least three, orall of TRP36R1, TRP140, TRP95R, and/or TRP95C. Each of the immunogenicsequences may be contiguously repeated 1-2 times. The polypeptide maycomprise TRP36R1, TRP140, TRP95R, and TRP95C. In some embodiments, thepolypeptide comprises or consists of SEQ ID NO:35. The polypeptide maycomprise or consist of a polypeptide of any one of (SEQ ID NOs: 25-35 or43-44). The isolated polypeptide may comprise 3, 4, 5, 6, 7, 8, 9, orall of the following immunogenic sequences: TRP120, A34N1, TRP63, TRP47,TRP75, TRP28, TRP36 (such as TRP36R1, TRP36R2, TRP36R3, and/or TRP36CO),TRP19, TRP140, and/or HSP. In some embodiments, the polypeptide furthercomprises at least two of TRP36R1, TRP36R2, TRP36R3, and/or TRP36CO. Thepolypeptide may comprise TRP36R1, TRP36R2, and TRP36R3. The TRP36R1,TRP36R2, and TRP36R3 sequences may be separated by a linker. In someembodiments, the TRP36R1, TRP36R2, and TRP36R3 sequences are notseparated by a linker. The polypeptide may comprise TRP36R1-R2-R3 (SEQID NO:11), TRP36R1-R3-R2 (SEQ ID NO:12), TRP36R2-R1-R3 (SEQ ID NO:13),TRP36R2-R3-R1 (SEQ ID NO:14), TRP36R3-R1-R2 (SEQ ID NO:15), orTRP36R3-R2-R1 (SEQ ID NO:16). In some embodiments, each of theimmunogenic sequences in the polypeptide are contiguously repeated 1, 2,or 3 times. Each of the immunogenic sequences may be contiguouslyrepeated 1 or 2 times. In some embodiments, the different immunogenicsequences are not separated by a linker or a spacer. In someembodiments, the different immunogenic sequences are separated by alinker or a spacer, such as for example a glycine linker. The glycinelinker may have the amino acid sequence -(G)x-, wherein X=3-5. In someembodiments, the polypeptide is less than 500, less than 450, less than400, less than 350, less than 300, less than 250, less than 200, or lessthan 150 amino acids in length.

In some embodiments, the polypeptide is comprised in a pharmaceuticalpreparation. In some embodiments, the pharmaceutical preparation isformulated for parenteral, intravenous, subcutaneous, intranasal,sublingual, or intradermal administration. In some embodiments, thepolypeptide is attached to a solid support (e.g., glass or plastic) orcomprised in a diagnostic kit. In some embodiments, the solid support iscomprised in a lateral flow assay, or microfluidic device.

Another aspect of the present invention relates to an isolatedpolypeptide of Formula I(A_(s)-B_(t)-C_(u)-D_(v)-E_(w)-F_(x)-G_(y)-H_(z))_(n), wherein A, B, C,D, E, F, G, and H is a peptide selected from SEQ ID NOs:1-24 and 36-42,or a sequence at least 90% identical (preferably at least 95% identical)to any one of (SEQ ID NOs:1-24 or 36-42), wherein s, t, u, v, x, y, andz is an integer 0-8, wherein at least two (e.g., 2, 3, 4, 5, 6, 7, or 8)of s-z are ≥1 and at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of s-zis ≥2, and wherein n is an integer 1-5. In some embodiments, A is SEQ IDNO:8 (TRP36R1) and B is SEQ ID NO:23 (TRP140). In some embodiments, s isfrom 4 to 8, t is from 2 to 4, and n=1. In some embodiments, u, v, x, y,and z are zero. In some embodiments, wherein s=8 and t=4. Thepolypeptide may comprise or consist of SEQ ID NO:27. In someembodiments, at least two, three, four, five or six of s, t, u, v, x, y,and z are each 2-3; and wherein n=1. In some embodiments, A is TRP32(e.g., TRP32R3 of SEQ ID NO:5), B is TRP120 (SEQ ID NO:22), C is TRP36R1(SEQ ID NO:8), D is TRP140 (SEQ ID NO:23), E is TRP28 (SEQ ID NO:2), andF is HSP (SEQ ID NO:24). In some embodiments, z=0. The polypeptide maycomprise or consist of SEQ ID NO:28. In some embodiments, thepolypeptide is a polypeptide of Table 2 or any one of (SEQ ID NOs: 25-35or 43-44).

Yet another aspect of the present invention relates to a pharmaceuticalpreparation comprising a polypeptide disclosed herein (e.g., of FormulaI or in Table 2) or as described above, and a pharmaceuticallyacceptable excipient. The pharmaceutical preparation may be formulatedfor parenteral, intravenous, subcutaneous, intranasal, sublingual, orintradermal administration. The pharmaceutically acceptable excipientmay comprise or consists of an adjuvant. In some embodiments, theadjuvant is an emulsion or liposomes, or wherein the adjuvant comprisesa lipid. The emulsion may be an oil-in-water (O/W) emulsion or awater-in-oil (W/O) emulsion. In some embodiments, the adjuvant comprisesa triterpenoid, a sterol, an immunomodulator, a polymer (e.g.,diethyl-aminoethyl (DEAE)-dextran, polyethelyne glycol, or polyacrylicacid), and/or an immunostimulatory oligonucleotide (e.g., a CpGcontaining ODN). In some embodiments, the adjuvant comprises DEAEDextran, an immunostimulatory oligonucleotide, and oil such as mineraloil, wherein the immunostimulatory oligonucleotide is a CpG containingODN, and wherein the adjuvant formulation is a water-in-oil (W/O)emulsion. In some embodiments, the adjuvant comprises a saponin, asterol, a quaternary ammonium compound, a polymer, and an ORN/ODN. Insome embodiments, the saponin is Quil A or a purified faction thereof,the sterol is cholesterol, the quaternary ammonium compound is dimethyldioctadecyl ammonium bromide (DDA), the polymer is polyacrylic acid, andthe ORN/ODN is a CpG. In some embodiments, the saponin is present in anamount of about 1 μg to about 5,000 μg per dose, the sterol is presentin an amount of about 1 μg to about 5,000 μg per dose, the quaternaryammonium compound is present in an amount of about 1 μg to about 5,000μg per dose, and the polymer is present in an amount of about 0.0001%v/v to about 75% v/v. In some embodiments, the adjuvant furthercomprises a glycolipid such as, e.g.,N-(2-deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyldodecanamideacetate. In some embodiments, the adjuvant comprises a triterpenoidsaponin, a sterol, a quaternary ammonium compound, and a polyacrylicacid polymer. In some embodiments, the saponin is Quil A or a purifiedfraction thereof, the sterol is cholesterol, and the quaternary ammoniumcompound is dimethyl dioctadecyl ammonium bromide (DDA). In someembodiments, the saponin is present in an amount of about 1 mg to about5,000 mg per dose, the sterol is present in an amount of about 1 mg toabout 5,000 mg per dose, the quaternary ammonium compound is present inan amount of about 1 mg to about 5,000 mg per dose, and the polyacrylicacid polymer is present in an amount of about 0.0001% v/v to about 75%v/v. The adjuvant may comprise a water-in-oil emulsion. The water-in-oilemulsion may comprise an oily phase and an aqueous phase, a polycationiccarrier (e.g., DEAE dextran), and a CpG containing immunostimulatoryoligonucleotide. In some embodiments, the composition further comprisesan aluminum hydroxide gel. In some embodiments, the polycationic carrieris DEAE dextran. The composition may comprise an emulsion or anoil-in-water (O/W) emulsion. In some embodiments, the emulsion comprisesan aqueous phase that comprises an alkyl-polyacrylic acid (alkyl-PAA) orboth an acrylic polymer and dimethyl dioctadecyl ammonium bromide (DDA).In some embodiments, the aqueous phase of the oil-in-water emulsioncomprises dimethyl dioctadecyl ammonium bromide (DDA) and analkyl-polyacrylic acid (alkyl-PAA). In some embodiments, the alkyl-PAAis decyl-PAA, octyl-PAA, butyl-PAA, or methyl-PA. In some embodiments,the acrylic polymer is a polymer of acrylic acid crosslinked withpolyallyl sucrose. The composition may comprise a water-in-oil (W/O)emulsion comprising a non-mineral oil and an emulsifier (e.g., a mannidemono-oleate emulsifier). In some embodiments, the adjuvant is MF59,AS01, AS02, AS03, AS04, Virosomes, CAF01, CAF04, CAF05, an acrylicpolymer/DDA emulsion, a CpG/DEAE emulsion, a saponin/cholesterol/DDAadjuvant, or a polyacrylic acid polymer emulsion. In some embodiments,the composition further comprises an Ehrlichia bacterin. The bacterincan be, e.g., a heat-inactivated E. canis, a chemically-inactivated E.canis, a heat-inactivated E. chaffeensis or a chemically-inactivated E.chaffeensis. In some embodiments, the bacterin is achemically-inactivated bacterin that has been inactivated withformaldehyde, formalin, bi-ethylene amine, radiation, ultraviolet light,beta-propiolactone treatment, or formaldehyde.

Another aspect of the present invention relates to a nucleic acidencoding a polypeptide provided herein (e.g., a polypeptide of Formula 1or in Table 2) or as described above. The nucleic acid may be a DNAsegment. The nucleic acid may be comprised in an expression vector.

Yet another aspect of the present invention relates to a host cellcomprising a nucleic acid provided herein (e.g., a polypeptide ofFormula 1 or in Table 2) or as described above. In some embodiments, thecell expresses the nucleic acid.

Another aspect of the present invention relates to a method of detectingantibodies that specifically bind an Ehrlichia organism in a testsample, comprising: (a) contacting an isolated polypeptide providedherein (e.g., a polypeptide of Formula 1 or in Table 2) or as describedabove; (b) detecting the peptide-antibody complexes; wherein thedetection of the peptide-antibody complexes is an indication thatantibodies specific for an Ehrlichia organism are present in the testsample, and wherein the absence of the peptide-antibody complexes is anindication that antibodies specific an Ehrlichia organism are notpresent in the test sample. The Ehrlichia organism may be an Ehrlichiachaffeensis organism or an Ehrlichia canis organism. The step ofdetecting may comprise performing an enzyme-linked immunoassay, aradioimmunoassay, an immunoprecipitation, a fluorescence immunoassay, achemiluminescent assay, an immunoblot assay, a lateral flow assay, aflow cytometry assay, a multiplex immunoassay, a mass spectrometryassay, or a particulate-based assay. The step of detecting may comprisea lateral flow assay or an enzyme-linked immunoassay, wherein theenzyme-linked immunoassay is an ELISA.

Yet another aspect of the present invention relates to a method ofidentifying an Ehrlichia infection in a mammalian subject comprising:(a) contacting a biological sample from the subject with an isolatedpolypeptide provided herein (e.g., a polypeptide of Formula 1 or inTable 2) or as described above under conditions that allowpeptide-antibody complexes to form; and (b) detecting thepeptide-antibody complexes; wherein the detection of thepeptide-antibody complexes is an indication that the subject has anEhrlichia infection. The step of detecting may comprise performing anenzyme-linked immunoassay, a radioimmunoassay, an immunoprecipitation, afluorescence immunoassay, a chemiluminescent assay, an immunoblot assay,a lateral flow assay, a flow cytometry assay, a multiplex immunoassay, adipstick test, or a particulate-based assay. In some embodiments, thesubject is a human or a dog.

Another aspect of the present invention relates to a kit comprising: (a)an isolated polypeptide disclosed herein or as described above (e.g., apolypeptide of Formula 1 or in Table 2), (b) an anti-dog or anti-humansecondary antibody linked to a reporter molecule; and, (c) anappropriate reagent for detection of the reporter molecule. The peptidemay be immobilized on a membrane or a microtiter plate. The reportermolecule may be selected from the group consisting of luciferase,horseradish peroxidase, a luminous nanoparticle, P-galactosidase, and afluorescent label. The luminous nanoparticle may be a strontiumaluminate nanoparticle. The kit may further comprise a dilution bufferfor dog or human serum. The kit may comprise a lateral flow immunoassayor a lateral flow immunochromatographic assay. In some embodiments, thekit comprises an enzyme-linked immunosorbent assay (ELISA).

Yet another aspect of the present invention relates to a method ofinducing an immune response in a mammalian subject comprisingadministering to the subject an effective amount of a pharmaceuticalpreparation comprising a polypeptide provided herein (e.g., apolypeptide of Formula 1 or in Table 2) or a pharmaceutical preparationas described above. The subject may be a human or a dog. Thepharmaceutical preparation may be administered subcutaneously,intramuscularly, nasally, via inhalation or aerosol delivery, orintradermally.

Another aspect of the present invention relates to a method of treatingan Ehrlichia chaffeensis or Ehrlichia canis infection in a subjectcomprising: (a) contacting a biological sample from the subject with anisolated polypeptide provided herein provided herein (e.g., apolypeptide of Formula 1 or in Table 2) or as described above underconditions that allow peptide-antibody complexes to form; (b) detectingthe peptide-antibody complexes; wherein the detection of thepeptide-antibody complexes is an indication that the subject has anEhrlichia chaffeensis or Ehrlichia canis infection; and (c)administering a therapeutic compound to treat Ehrlichia infection in thesubject. The step of detecting may comprise performing an enzyme-linkedimmunoassay, a radioimmunoassay, an immunoprecipitation, a fluorescenceimmunoassay, a chemiluminescent assay, an immunoblot assay, a lateralflow assay, a flow cytometry assay, a multiplex immunoassay, a dipsticktest, or a particulate-based assay. The subject may be a dog or a human.The therapeutic compound may be an antibiotic (e.g., doxycycline).

As used herein, the term “contiguously repeated”, when used to describea nucleic acid sequence or amino acid sequence, indicates that thesequence is repeated in a polypeptide from an N-terminus to C-terminusdirection. For example, for amino acid sequence -X₁-, if the sequence isrepeated zero times, then it is included in the polypeptide withoutrepetition as -X₁-. If the sequence -X₁- is contiguously repeated once,then the polypeptide contains -X₁-X₁-. If the sequence -X₁- iscontiguously repeated twice then the polypeptide contains -X₁-X₁-X₁-.The number of contiguous repetitions of the sequence -X₁- may bedescribed by the formula -(X₁)_((n+1))-, wherein (n+1) refers to thenumber of contiguous repetitions. Preferably, the contiguously repeatedsequences are repeated without any linker or spacer sequence separatingthe contiguously repeated sequences. Nonetheless, in some embodiments, aspacer or linker (e.g., a glycine linker such as -G-, -GG-, or -GGG-)may be used to separate the contiguously repeated sequences. In somepreferred embodiments, different contiguously repeated sequences areseparated by a spacer or linker (e.g., a glycine linker such as -G-,-GG-, or -GGG-); for example, in sequence -X₁-X₁-GG-X₂-X₂-X₂-, sequence-X₁- is contiguously repeated once and sequence -X₂- is contiguouslyrepeated twice, wherein the different contiguously repeated sequencesare separated by the glycine linker -GG-.

As used herein, the term “polypeptide” encompasses amino acid chainscomprising at least 50 amino acid residues, and more preferably at least100 amino acid residues, wherein the amino acid residues are linked bycovalent peptide bonds. As used herein, an “antigenic polypeptide” or an“immunoreactive polypeptide” is a polypeptide which, when introducedinto a vertebrate, can stimulate the production of antibodies in thevertebrate, i.e., is antigenic, and wherein the antibody can selectivelyrecognize and/or bind the antigenic polypeptide. An antigenicpolypeptide may comprise or consist of an immunoreactive sequence(s)derived from an immunoreactive Ehrlichia protein as described herein;for example, polypeptides in Table 1, Table 2, and Formula I), and thepolypeptide may comprise one or more additional sequences. In someembodiments, the additional sequences may be derived from a nativeEhrlichia antigen and may be heterologous, and such sequences may (butneed not) be immunogenic. In some embodiments, the antigenic polypeptideor immunoreactive polypeptide may be covalently bound to a solidsubstrate, e.g., in an immunoassay such as a lateral flow test, etc.

Ehrlichia immunoreactive polypeptides as described herein (e.g., inTable 2 or Formula I) may be a recombinant polypeptide, syntheticpolypeptide, purified polypeptide, immobilized polypeptide, detectablylabeled polypeptide, encapsulated polypeptide, or a vector-expressedpolypeptide. In various embodiments, the Ehrlichia immunoreactivepolypeptides provided herein may be truncated or may comprise a deletionmutation, without eliminating the immunoreactivity of the resultingpeptide or polypeptide. An immunoreactive peptide or polypeptidedisclosed herein may also be comprised in a pharmaceutical compositionsuch as, e.g., a vaccine composition that is formulated foradministration to a human or canine subject.

“Bacterin” as used herein refers to one or more killed bacteria whichmay be used as a component of a vaccine or immunogenic composition. Thebacterin may be comprised in a suspension. In some preferredembodiments, the bacterin is a heat-inactivated Ehrlichia (e.g., aheat-inactivated E. canis) or a chemically-inactivated Ehrlichia (e.g.,a chemically-inactivated E. Canis).

“Adjuvant” as used herein refers to any substance that increases thehumoral or cellular immune response to an antigen. In some embodiments,Adjuvants be used to both allow for the controlled release of antigensfrom the injection site of a vaccine and stimulate the immune system ofthe subject receiving the vaccine composition.

As used herein, “essentially free,” in terms of a specified component,is used herein to mean that none of the specified component has beenpurposefully formulated into a composition and/or is present only as acontaminant or in trace amounts. The total amount of the specifiedcomponent resulting from any unintended contamination of a compositionis preferably below 0.01%. Most preferred is a composition in which noamount of the specified component can be detected with standardanalytical methods.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation or error of the device useddetermine the value, the method employed to determine the value, or thevariation that exists among the study subjects or samples.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1: Purification and Characterization of E. chaffeensis chimeraTRP32/TRP120/A34 (Combined=SEQ ID NO: 25). TRP32R1=SEQ ID NO: 3;TRP32R2=SEQ ID NO: 4; TRP32R3=SEQ ID NO: 5; TRP32R4=SEQ ID NO: 6;TRP120=SEQ ID NO: 22; A34N1=SEQ ID NO: 7.

FIG. 2: Purification and Characterization of E. chaffeensis chimeraTRP140/TRP36/TRP19 (Combined=SEQ ID NO: 26). TRP19=SEQ ID NO: 1;TRP36=SEQ ID NO: 8; TRP140=SEQ ID NO: 23.

FIG. 3: Purification and Characterization of E. canis chimeraTRP36/TRP140 (Combined=SEQ ID NO: 27). TRP36=SEQ ID NO: 8; TRP140=SEQ IDNO: 23.

FIG. 4: Purification and Characterization of E. chaffeensis and E. canischimera TRP32/TRP120/TRP36/TRP140/HSP (Combined=SEQ ID NO: 28).TRP32=SEQ ID NO: 5; TRP120=SEQ ID NO: 22; TRP36=SEQ ID NO: 8; TRP140=SEQID NO: 23; P28=SEQ ID NO: 2; HSP=SEQ ID NO: 24.

FIG. 5: Purification and Characterization of E. chaffeensis and E. canischimera TRP120/TRP140/TRP36/TRP28 (Combined=SEQ ID NO: 29). TRP120=SEQID NO: 22; TRP140=SEQ ID NO: 23; TRP36=SEQ ID NO: 8; P28=SEQ ID NO: 2.

FIG. 6: Additional Ehrlichia chimeric polypeptides.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In some embodiments, an immunoreactive polypeptide (e.g., a polypeptideof Formula I or a polypeptide in Table 2) described herein may be usedas diagnostic or prophylactic tools for detection of or immunizationagainst Ehrlichia infection. In particular embodiments, immunoreactivepolypeptides disclosed herein may be useful in solution-phase assays, orin assays in which the isolated immunoreactive polypeptide isimmobilized on a surface of a support substrate. Alternatively, animmunoreactive polypeptide described herein may be comprised in avaccine formulation to induce a protective immune response or an immuneresponse against E. chaffeensis in a subject. One or more immunoreactivepolypeptides as described herein may be immobilized on a surface bycovalent attachment, encapsulation, or adsorption using methodsgenerally known in the art, and may include the use of cross-linkers,capture molecules and such like, to which peptides may be coupled,conjugated, or cross-linked.

I. CHIMERIC POLYPEPTIDES

As described in the foregoing summary, certain aspects of the presentdisclosure concern chimeric Ehrlichia polypeptides, such as polypeptidescomprising or consisting of at least two of the peptides of Table 1 or apolypeptide of Table 2. The chimeric polypeptides may be produced tocomprise at least two of the peptides of Table 1 which can becontinuously repeated (e.g., from 2 to 8, preferably 2 to 6, or evenmore preferably 2 to 4 times) in the polypeptide. The chimericpolypeptide can comprise 3, 4, 5 or more of the above immunogenicpeptides. Each of the immunogenic peptides may be repeated 0, 1, 2, 3,4, 5, 6, 7, or 8 times in the chimeric polypeptide. Exemplaryconfigurations include, but are not limited to, 2×2, 2×2×2, 2×2×2×2,3×2, 2×3, 3×2×2, 2×3×2, 2×2×3, 3×2×2×2, 2×3×2×2, 2×2×3×2, 2×2×2×3, 3×3,3×3×3, 3×3×2, 2×2×3, 3×3×3×3, 3×3×3×6, and 2×2×4×2×2×2.

TABLE 1 Ehrlichia immunogenic peptides. Immunogenic Peptide SequenceSpecies TRP19 HFTGPTSFEVNLSEEEKMELQEVS SEQ ID E. canis NO: 1 P28AKEEKNATAKTFQLKGDWDGA SEQ ID E. canis NO: 2 TRP32R1SDLHESSFVELPGPSKEEVQFEDDAKNVVY SEQ ID E. NO: 3 chaffeensis TRP32R2SDLHGSFSVELFDPSKEEVQLESDLQQSSN SEQ ID E. NO: 4 chaffeensis TRP32R3SDLHGSFSVELFDPFKEAVQLGNDLQQSSD SEQ ID E. NO: 5 chaffeensis TRP32R4SDSHEPSHLELPSLSEEVIQLESDLQQSSN SEQ ID E. NO: 6 chaffeensis A34N1VRSITDPRIVVQQEADQQQEVQQQAD SEQ ID E. NO: 7 chaffeensis TRP36R1 TEDSVSAPASEQ ID E. canis NO: 8 TRP36R2 ASVVPEAE SEQ ID E. canis NO: 9 TRP36R3TEDPVSATA SEQ ID E. canis NO: 10 TRP36R1- TEDSVSAPA ASVVPEAE TEDPVSATASEQ ID R2-R3 NO: 11 TRP36R1- TEDSVSAPA TEDPVSATA ASVVPEAE SEQ ID R3-R2NO: 12 TRP36R2- ASVVPEAE TEDSVSAPA TEDPVSATA SEQ ID R1-R3 NO: 13TRP36R2- ASVVPEAE TEDPVSATA TEDSVSAPA SEQ ID R3-R1 NO: 14 TRP36R3-TEDPVSATA TEDSVSAPA ASVVPEAE SEQ ID R1-R2 NO: 15 TRP36R3-TEDPVSATA ASVVPEAE TEDSVSAPA SEQ ID R2-R1 NO: 16 TRP47ASVSEGDAVVNAVSQETPA SEQ ID E. NO: 17 chaffeensis TRP63SLFTEEEKILAILSARFICK SEQ ID E. NO: 18 chaffeensis TRP75DVKDNKPSDVKLPVIKAE SEQ ID E. NO: 19 chaffeensis TRP95RDDSKLPVIKVEDKSKLQDTKDKKR SEQ ID E. canis NO: 20 TRP95CKKIKEYDEDYTITYYYDDD SEQ ID E. canis NO: 21 TRP120 SKVEQEETNPEVLIKDLQDVASSEQ ID E. NO: 22 chaffeensis TRP140 EHSSSEVGEKVSETSKEENTPEVKA SEQ IDE. canis NO: 23 HSP YGAPEITKDGYKVIKSIKPED SEQ ID E. NO: 24 chaffeensisTRP36CO EASVVPAAEAPQPAQQTEDEFFSDGIEA SEQ ID E. canis NO: 36 TRP36CO-EASVVPAAEAPQPAQQTEDEFFSDGIEA SEQ ID R1 TEDSVSAPA NO: 37 TRP36R1-TEDSVSAPA SEQ ID CO EASVVPAAEAPQPAQQTEDEFFSDGIEA NO: 38 TRP36CO-EASVVPAAEAPQPAQQTEDEFFSDGIEA SEQ ID R3 TEDPVSATA NO: 39 TRP36R3-TEDPVSATA SEQ ID CO EASVVPAAEAPQPAQQTEDEFFSDGIEA NO: 40 TRP36R1-R3TEDSVSAPA TEDPVSATA SEQ ID NO: 41 TRP36R3-R1 TEDPVSATA TEDSVSAPA SEQ IDNO: 42

In some embodiments, it is anticipated that TRP36R1, TRP36R2, andTRP36R3 may be switched within a chimeric polypeptide. For example, insome embodiments, a TRP36R1 sequence in a chimeric polypeptide may beswitched for TRP36R2 or TRP36R3. In some embodiments, a TRP36R1,TRP36R2, or TRP36R3 sequence in a chimeric polypeptide may be switchedfor TRP36CO. In some embodiments, it has been observed that TRP36R1,TRP36R2, TRP36R3, and/or TRP36CO may be contiguously located in achimeric polypeptide, e.g., as shown in SEQ ID NOs:11-16 and 38-43. Insome embodiments, 2, 3, or all of TRP36R1, TRP36R2, TRP36R3, and/orTRP36CO may be contiguously located in a chimeric polypeptide.Similarly, it is anticipated that, in some embodiments, TRP32R1,TRP32R2, TRP32R3, and TRP32R4 may be switched within a chimericpolypeptide. In some embodiments, 2, 3, or all of TRP32R1, TRP32R2,TRP32R3, and/or TRP32R4 may be contiguously located in a chimericpolypeptide (e.g., wherein the polypeptide contains-TRP32R1-TRP32R2-TRP32R3- or -TRP32R1-TRP32R2-TRP32R3-TRP32R4-, etc.).

The chimeric polypeptides may be produced to comprise at least two ofthe above peptides which can be continuously repeated (e.g., once, from2 to 8, preferably 2 to 6, or even more preferably 2 to 4, times) in thepolypeptide. The chimeric polypeptide can comprise 3, 4, 5 or more ofthe above immunogenic peptides from Table 1. Each of the immunogenicpeptides may be repeated 1, 2, 3, 4, 5, 6, 7, or 8 times in the chimericpolypeptide. The peptides within the polypeptide construct may beseparated by a linker. The linker can be a poly-glycine linker (e.g.,GG, GGG, GGGG, or GGGGG). The polypeptide constructs may comprise a heatshock protein (HSP) sequence (SEQ ID NO:24). Constructs were producedfrom the above immunogenic peptides as listed below (e.g., Table 2) andexemplary chimeric polypeptides are depicted in FIGS. 1-5.

In some embodiments, the chimeric polypeptide may comprise at least twoor three immunogenic peptide isoforms. For example, the polypeptide maybe produced by linking TRP36R1, TRP36R2, TRP36R3, and/or TRP36CO invarious configurations as listed in Table 1 including TRP36R1-R2-R3,TRP36R1-R3-R2, TRP36R2-R1-R3, TRP36R2-R3-R1, TRP36R3-R1-R2,TRP36R3-R2-R1, TRP36CO-R1, TRP36R1-CO, TRP36CO-R3, TRP36R3-CO,TRP36R1-R3, and TRP36R3-R1. The polypeptide may be produced by linkingTRP95R and TRP95C as TRP95R-C or TRP95C-R. In some embodiments, thepolypeptide may be produced by linking TRP32R1, TRP32R1, TRP32R3, and/orTRP32R4 in various configurations.

In some embodiments, the chimeric construct may have a sequence asdescribed by Formula I, below:

(A_(s)-B_(t)-C_(u)-D_(v)-E_(w)-F_(x)-G_(y)-H_(z))_(n),  Formula I:

wherein A-H comprise a peptide selected from SEQ ID NOs:1-24 and 36-42,s-z is an integer 0-8, wherein at least two of s-z are ≥1 and at leastone of s-z is ≥2, and n is an integer 1-5. Peptides A-H may be each beindependently selected from SEQ ID NOs:1-24 and 36-42. Preferably, thechimeric construct may have a sequence of Formula I, wherein at leasttwo of s-z are ≥2. The chimeric construct may comprise a linker orspacer to separate the peptides A-H of Formula I.

Exemplary derivatives of Formula I may comprise, but are not limited to:

A₂-B₂-C₂;

A₁-B₃-C₃;

A₃-B₃-C₃;

A₂-B₂-C₂-D₂;

A₃-B₂-C₂-D₂;

A₃-B₃-C₆-D₃;

(A₁-B₂-C₁)₃;

A₈-B₄; and

A₂-B₂-C₄-D₂-E₂-F₂.

In some embodiments, the polypeptide of Formula I comprises or consistsof any one of SEQ ID NOs: 11-16 or 38-42. In some embodiments, thepolypeptide of Formula I comprises or consists of a polypeptide of Table2.

TABLE 2 Chimeric Ehrlichia Polypeptides Construct Sequence SpeciesTRP32/TRP120/A34N1 SEQ ID NO: 25 E. chaffeensis (32R1/32R2/32R3/32R4/3 ×120/3 × 43) TRP140/TRP36/TRP19 SEQ ID NO: 26 E. canis (19/2 × 36/140) ×3 TRP36/TRP140 SEQ ID NO: 27 E. canis (8 × 36/4 × 140)TRP32/TRP120/TRP36/TRP140/TRP28/HSP SEQ ID NO: 28 E. chaffeensis and (2× 32/2 × 120/4 × 36/2 × 140/2 × 28/2 × HSP) E. canisTRP120/TRP140/TRP36/TRP28 SEQ ID NO: 29 E. chaffeensis and (3 × 120/3 ×140/6 × 36/3 × 28) E. canis TRP120/A34N1/TRP63/TRP47 SEQ ID NO: 30 E.chaffeensis (2 × 120/2 × 34/2 × 63/2 × 47) TRP120/A34N1/TRP63 SEQ ID NO:31 E. chaffeensis (2 × 120/2 × 34/2 × 63) TRP120/A34N1/TRP47 SEQ ID NO:32 E. chaffeensis (2 × 120/2 × 34/2 × 63/2 × 47) A34N1/TRP63/TRP47 SEQID NO: 33 E. chaffeensis (2 × 34/2 × 63/2 × 47) A34N1/TRP63/TRP75 SEQ IDNO: 34 E. chaffeensis (2 × 34/2 × 63/2 × 75) TRP36/TRP140/TRP95R/TRP95CSEQ ID NO: 35 E. canis (2 × 36/2 × 140/2 × 95R/2 × 95C)TRP36R1/TRP36R2/TRP36CO SEQ ID NO: 43 E. canis (2 × 36/2 × 36/2 × 36)TRP36R1/TRP36R2 SEQ ID NO: 44 E. canis (2 × 36/2 × 36)

In some embodiments, it is anticipated that a polypeptide having atleast 90%, more preferably at least 95%, 97.5%, or at least 99% sequenceidentity to a polypeptide of Table 2 or a polypeptide of Formula I, thatretains at least some of its immunoreactivity may be used in variousembodiments as described herein (e.g., in a diagnostic test, or toinduce an immune response against Ehrlichia in a subject, for inclusionin a vaccine composition). In some embodiments, the polypeptide may beused to generate an antibody that selectively binds the protein, and theantibody may be used, e.g., in a diagnostic assay; for example, in someembodiments, the antibody is labelled or attached to a solid substrate(e.g., in a lateral-flow test).

In some aspects, a contiguous repeat of a specific peptide may comprisetwo or more isoforms of the immunogenic peptide. For example, acontiguous repeat may comprise TRP32R1, TRP32R2, TRP32R3, and/orTRP32R4. Another contiguous repeat can comprise TRP95C and TRP95R ordifferent isoforms of TRP36 including TRP36R1, TRP36R2, TRP36R3, and/orTRP36CO. Thus, the same or different isoforms of the protein may thus becontiguously repeated in some embodiments without separation by a spaceror glycine spacer.

A variety of linkers or spacers can be used in chimeric Ehrlichiapolypeptides of the embodiments. In some embodiments, a linker or spacercan be a random string of one or more amino acids (e.g., 2, 3, 4, 5, 10,15, or 20 amino acids). In other embodiments, the linker has aparticular sequence. For example, in some embodiments, the linker orspacer can be a poly-glycine linker (e.g., GG, GGG, or GGGG; alsoreferred to as a glycine linker herein). Other linkers or spacers thatmay be used in various embodiments include, e.g., the 218(GSTSGSGKPGSGEGSTKG; SEQ ID NO: 45), the HL (EAAAK; SEQ ID NO: 46), andthe G₄S (GGGGS; SEQ ID NO: 47) linkers. In some preferred embodiments,groups of different contiguously repeated sequences are separated in apolypeptide by a linker or spacer such as, e.g., a glycine linker (e.g.,-X₁-X₁-X₁-GGG-X₂-X₂- shows amino sequence -X₁- contiguously repeatedtwice, amino acid sequence -X₂- is contiguously repeated once, and thedifferent contiguously repeated sequences are separated by thepoly-glycine linker -GGG-).

In some embodiments, an immunoreactive polypeptide provided herein(e.g., derived from two or more peptides in Table 1 or a polypeptide inTable 2) may be immobilized onto a surface of a support or a solidsubstrate; for example, the immunoreactive polypeptide may beimmobilized directly or indirectly by coupling, cross-linking,adsorption, encapsulation, or by any appropriate method known in theart. By way of non-limiting example, binding of an immunoreactivepolypeptide disclosed herein by adsorption to a well in a microtiterplate or to a membrane may be achieved by contacting the peptide, in asuitable buffer, with the well surface for a suitable amount of time.The contact time can vary with temperature, but is typically betweenabout 1 hour and 1 day when using an amount of peptide ranging fromabout 50 ng to about 1 mg, and preferably about 250-700 ng or about450-550 ng.

In some embodiments, an immunoreactive polypeptide disclosed herein iscovalently attached to a support substrate by first reacting the supportwith a reagent that will chemically react with both the support and afunctional group (i.e., crosslink), such as a hydroxyl or amino group,on the peptide. For example, an immunoreactive polypeptide may becrosslinked to a surface through an amine or carboxylic group on eitherend of the peptide, and a peptide may be crosslinked through a group oneach end of the polypeptide (i.e., head-to-tail crosslinked). Suchpeptomers (i.e., head-to-tail crosslinked or otherwise immobilizedpeptides) may be used with both diagnostic and therapeutic methods ofthe present disclosure.

In some embodiments, an isolated polypeptide comprising a sequence of apolypeptide of Formula I or a polypeptide Table 2, wherein the isolatedpeptide or polypeptide is immobilized on a surface of a supportsubstrate. In some embodiments, the polypeptide is selected from thegroup consisting of Table 2. Numerous support substrates for peptideimmobilization are known in the art which may be employed with animmunoreactive polypeptide disclosed herein, formed from materials suchas, for example, latex, polystyrene, nylon, nitrocellulose, cellulose,silica, agarose, inorganic polymers, lipids, proteins, sugars, ormagnetic resin. A person of ordinary skill in the art may select thesupport substrate that is appropriate for a given application. Inparticular embodiments, a support substrate may be a reaction chamber, amicroplate well, a membrane, a filter, a paper, an emulsion, a bead, amicrobead, a microsphere, a nanocrystal, a nanosphere, a dipstick, acard, a glass slide, a microslide, a lateral flow apparatus, amicrochip, a comb, a silica particle, a magnetic particle, ananoparticle, or a self-assembling monolayer.

II. DETECTABLY-LABELED IMMUNOREACTIVE POLYPEPTIDES

An immunoreactive polypeptide (e.g., comprising two or more peptide ofTable 1, or a polypeptide of Table 2) may be conjugated to or attachedto detectable label such as, for example, a radioactive isotope, anon-radioactive isotope, a particulate label, a fluorescent label, achemiluminescent label, a paramagnetic label, an enzyme label or acolorimetric label. The detectably-labelled polypeptide may be used,e.g., in diagnostic or prophylactic methods and compositions. In certainembodiments, the polypeptide portion of the detectably labeledimmunoreactive polypeptide may be immobilized on a surface of a supportsubstrate. In other embodiments, the detectable label may be used toimmobilize the detectably labeled immunoreactive peptide to the surfaceof a support substrate.

As used herein, “detectable label” is a compound and/or element that canbe detected due to its specific functional properties, and/or chemicalcharacteristics, the use of which allows the peptide to which it isattached be detected, and/or further quantified if desired.

In some embodiments, the probe is a photoluminescent probe, such as afluorophore or a nanoparticle, such as for example a strontium aluminatenanoparticle (e.g., see Paterson et al., 2014). Exemplary labelsinclude, but are not limited to, a particulate label such as colloidalgold, a radioactive isotope such as astatine²¹¹, ¹⁴carbon, ⁵¹chromium,³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen,iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus,rhenium186, rhenium188, ⁷⁵selenium, ³⁵sulphur, technicium-99,technetium-99m or yttrium⁹⁰, a colorimetric label such asdinitrobenzene, dansyl chloride, dabsyl chloride, any of the azo, cyaninor triazine dyes, or chromophores disclosed in U.S. Pat. Nos. 5,470,932,5,543,504, or 6,372,445, all of which are incorporated herein byreference; a paramagnetic label such as chromium (III), manganese (II),iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) or erbium (III), afluorescent label such as Alexa 350, Alexa 430, AMCA, BODIPY 630/650,BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, CascadeBlue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, OregonGreen 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG,Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET,Tetramethylrhodamine, and/or Texas Red, or Lucifer Yellow, an enzymelabel such as urease, luciferase, alkaline phosphatase, (horseradish)hydrogen peroxidase, or glucose oxidase, or a chemiluminescent labelsuch as luminol, phthalazinedione, and others disclosed in any of U.S.Pat. Nos. 4,373,932, 4,220,450, 5,470,723, and U.S. Patent Application2007/0264664, all of which are incorporated herein by reference.

III. METHODS OF PRODUCING AN IMMUNOREACTIVE POLYPEPTIDE

An immunoreactive polypeptide as described herein may be produced usingin vitro transcription and translation (IVTT) methods, may berecombinantly produced using a variety of cell types (e.g., bacterialcells, mammalian cells, E. coli, yeast, and insect cells, etc.), or insome instances may be synthesized (e.g., using solid-phase synthesis).In some embodiments, IVTT and synthetic methods can provide certainadvantages over recombinant approaches, since the resulting polypeptidescan produced highly pure forms without contaminating bacterial or otherproteins that might result in false positive reactions when utilizingrecombinant proteins. Thus, IVTT and synthetic methods have an advantageof lacking many of the costly and laborious purification proceduresoften associated with recombinant methodologies.

A variety of IVTT approaches are known in the art and may be used invarious embodiments. IVTT generally involves cell-free methods forproduction or synthesis of a protein from DNA. The cell-free system forprotein production may use, e.g., E. coli extract, protozoan extracts,yeast extracts, human cell extract, wheat germ extract, mammalianextracts, extracts from cultured human cell lines, rabbit reticulocytelysate, insect cell extract, or reconstituted and purified E. colicomponents. A variety of kits are commercially available including,e.g., RTS (FivePrime, San Francisco, Calif.), Expressway™ (LifeTechnologies); S30 T7 high yield (Promega), One-step human IVT (ThermoScientific), WEPRO® (CellFree Sciences), TNT® coupled (Promega), RTSCECF (5 PRIME), TNT® Coupled (Promega), Retic lysate IVT™ (LifeTechnologies); TNT® T7 (Promega), EasyXpress Insect kit(Qiagen/RiN A),PURExpress® (New England Biolabs), and PURESYSTEM® (BioComber). Suchmethods can be used to incorporate unnatural amino acids into proteins,if desired. Cell-free expression systems that may be used in variousembodiments are also described, e.g., in Zemella et al., 2015.

An isolated immunoreactive protein as disclosed herein may be producedin some embodiments using an appropriate method known in the organicchemistry arts. For example, peptides may be produced using one of theestablished solid-phase peptide synthesis techniques that are well knownin the art. In some embodiments, peptides may be synthesized usingequipment for automated peptide synthesis that is widely available fromcommercial suppliers such as Perkin Elmer (Foster City, Calif.), or thepeptide may be chemically synthesized using solution-phase techniquessuch as those described in Carpino et al., 2003 or U.S. PatentApplication 2009/0005535. In some embodiments, the peptides or shorterproteins may be synthesized, e.g., using solid-phase peptide synthesis(SPPS), t-Boc solid-phase peptide synthesis, or Fmoc solid-phase peptidesynthesis.

In some embodiments, an immunoreactive protein as described herein canbe recombinantly prepared from a nucleic acid encoding the peptide. Sucha nucleic acid may be operably linked to an expression vector. By way ofnonlimiting example, an immunoreactive protein may be expressed from avector and isolated from the growth media of a host cell comprising thevector. In some embodiments, the immunoreactive protein may be producedin a cell-free system from a nucleic acid encoding the peptide.

An immobilized immunoreactive protein as disclosed herein may beconjugated, crosslinked, or adsorbed, either directly or indirectly ontoa surface of a support substrate. In some embodiments, an immobilizedimmunoreactive protein or peptide may be synthesized onto a supportsubstrate.

It is anticipated that virtually any method of protein or peptideimmobilization known in the art which would not impact the structure orfunction of the disclosed peptides may be used to immobilize animmunoreactive protein or peptide as disclosed herein. For example,peptide immobilization may be accomplished using a crosslinking orconjugation agent such as methyl-p-hydroxybenzimidate,N-succinimidyl-3-(4-hydroxyphenyl)propionate, using sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sSMCC),N-[maleimidocaproyloxy] sulfosuccinimide ester (sEMCS),N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), glutaraldehyde,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI),Bis-diazobenzidine (BDB), or N-acetyl homocysteine thiolactone (NAHT),and others disclosed in any of U.S. Pat. Nos. 5,853,744, 5,891,506,6,210,708, 6,617,142, 6,875,750, 6,951,765, 7,163,677, and 7,282,194,each incorporated herein by reference. Immunoreactive proteins may beconjugated directly or indirectly to any of the commercially availablesupport substrates having a surface coatings comprising crosslinkers,coupling agents, thiol or hydroxyl derivatizing agents, carboxyl- oramine-reactive groups such as of maleic anhydride (e.g., PierceImmunotechnology Catalog and Handbook, at A12-A13, 1991).

In some embodiments, a protein of the invention may also be immobilizedusing metal chelate complexation, employing, for example, an organicchelating agent such a diethylenetriaminepentaacetic acid anhydride(DTPA); EDTA; N-chloro-p-toluenesulfonamide; and/ortetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody (U.S.Pat. Nos. 4,472,509 and 4,938,948, each incorporated herein byreference). Proteins and peptides can also be immobilized by coupling toother peptides or to condensation groups immobilized on a surface orpresent in an immobilization buffer such as glutaraldehyde or periodate.Conjugates with fluorescence markers may also prepared in the presenceof such agents or by reaction with an isothiocyanate. A peptide may beattached to a surface by conjugation, crosslinking or binding to anaffinity binding agent such as biotin, streptavidin, a polysaccharidesuch as an alginate, a lectin, and the like.

In general, regardless of the method of preparation or immobilizationstatus, the immunoreactive proteins disclosed herein are preferablyprepared in a substantially pure form. Preferably, the immunoreactiveproteins are at least about 80% pure, more preferably at least about 90%pure and most preferably at least about 99% pure.

IV. BIOLOGICAL FUNCTIONAL EQUIVALENTS

Preferred immunoreactive polypeptides or analogs thereof specifically orpreferentially bind an E. chaffeensis or E. canis specific antibody.Determining whether or to what degree a particular immunoreactivepolypeptide, or an analog thereof, can bind an E chaffeensis or E. canisspecific antibody can be assessed using an in vitro assay such as, forexample, an enzyme-linked immunosorbent assay (ELISA), immunoblotting,immunoprecipitation, radioimmunoas say (RIA), immunostaining, latexagglutination, indirect hemagglutination assay (IHA), complementfixation, indirect immnunofluorescent assay (FA), nephelometry, flowcytometry assay, chemiluminescence assay, lateral flow immunoassay,u-capture assay, mass spectrometry assay, particle-based assay,inhibition assay and/or an avidity assay.

An immunoreactive polypeptide of the present disclosure may be modifiedto contain amino acid substitutions, insertions and/or deletions that donot alter their respective interactions with anti-Ehrlichia antibodybinding regions. Such a biologically functional equivalent of animmunoreactive polypeptide derived from an Ehrlichia protein could be amolecule having like or otherwise desirable characteristics, i.e.,binding of Ehrlichia specific antibodies. As a nonlimiting example,certain amino acids may be substituted for other amino acids in animmunoreactive polypeptide disclosed herein without appreciable loss ofinteractive capacity, as demonstrated by detectably unchanged antibodybinding. It is thus contemplated that an immunoreactive polypeptidedisclosed herein (or a nucleic acid encoding such a polypeptide) whichis modified in sequence and/or structure, but which is unchanged inbiological utility or activity, remains within the scope of the presentdisclosure. The immunoreactive polypeptide may have, e.g., at least 90%,95%, or 99% sequence identity with a wild-type E. chaffeensispolypeptide, and in some embodiments the immunoreactive protein may have1, 2, 3, 4, 5, or more amino acid substitutions, insertions and/ordeletions as compared with the corresponding wild-type E. chaffeensispolypeptide. In some embodiments, the mutation is a conservativesubstitution.

It is also well understood by the skilled artisan that, inherent in thedefinition of a biologically functional equivalent peptide, is theconcept that there is a limit to the number of changes that may be madewithin a defined portion of the molecule while still maintaining anacceptable level of equivalent biological activity. Biologicallyfunctional equivalent polypeptides are thus defined herein as thosepeptides in which certain, not most or all, of the amino acids may besubstituted. Of course, a plurality of distinct peptides with differentsubstitutions may easily be made and used in accordance with theinvention.

The skilled artisan is also aware that where certain residues are shownto be particularly important to the biological or structural propertiesof a peptide, e.g., residues in specific epitopes, such residues may notgenerally be exchanged. It is anticipated that a mutation in animmunoreactive peptide or polypeptide disclosed herein could result in aloss of species-specificity and in turn, reduce the utility of theresulting peptide for use in methods for generating an anti-Ehrlichiaimmune response. Thus, polypeptides which are antigenic (i.e., bindanti-Ehrlichia antibodies specifically) and comprise conservative aminoacid substitutions are understood to be included in aspects of thepresent disclosure. Conservative substitutions are least likely todrastically alter the activity of a protein. A “conservative amino acidsubstitution” refers to replacement of amino acid with a chemicallysimilar amino acid, i.e., replacing nonpolar amino acids with othernonpolar amino acids; substitution of polar amino acids with other polaramino acids, acidic residues with other acidic amino acids, etc.

Amino acid substitutions, such as those which might be employed inmodifying an immunoreactive polypeptide disclosed herein are generallybased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. An analysis of the size, shape and type of the aminoacid side-chain substituents reveals that arginine, lysine and histidineare all positively charged residues; that alanine, glycine and serineare all a similar size; and that phenylalanine, tryptophan and tyrosineall have a generally similar shape. Therefore, based upon theseconsiderations, arginine, lysine and histidine; alanine, glycine andserine; and phenylalanine, tryptophan and tyrosine; are defined hereinas biologically functional equivalents.

The invention also contemplates isoforms of the E. chaffeensisimmunoreactive polypeptides disclosed herein. An isoform contains thesame number and kinds of amino acids as an E. chaffeensis polypeptide asdisclosed herein, but the isoform has a different molecular structure.The isoforms contemplated by the present disclosure are those having thesame properties as a peptide of the invention as described herein.

Nonstandard amino acids may be incorporated into proteins by chemicalmodification of existing amino acids or by de novo synthesis of apolypeptide disclosed herein. A nonstandard amino acid refers to anamino acid that differs in chemical structure from the twenty standardamino acids encoded by the genetic code, and a variety of nonstandardamino acids are well known in the art.

In select embodiments, the present disclosure contemplates a chemicalderivative of an immunoreactive polypeptide disclosed herein. “Chemicalderivative” refers to a peptide having one or more residues chemicallyderivatized by reaction of a functional side group, and retainingbiological activity and utility. Such derivatized polypeptides include,for example, those in which free amino groups have been derivatized toform specific salts or derivatized by alkylation and/or acylation,p-toluene sulfonyl groups, carbobenzoxy groups, t-butylocycarbonylgroups, chloroacetyl groups, formyl or acetyl groups among others. Freecarboxyl groups may be derivatized to form organic or inorganic salts,methyl and ethyl esters or other types of esters or hydrazides andpreferably amides (primary or secondary). Chemical derivatives mayinclude polypeptides that comprise one or more naturally occurring aminoacids derivatives of the twenty standard amino acids. For example,4-hydroxyproline may be substituted for serine; and ornithine may besubstituted for lysine.

It should be noted that all amino-acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid residues. The amino acids describedherein are preferred to be in the “L” isomeric form. However, residuesin the “D” isomeric form can be substituted for any L-amino acidresidue, as long as the desired functional properties set forth hereinare retained by the protein. In keeping with standard proteinnomenclature, abbreviations for amino acid residues are known in theart.

In addition to the biological functional equivalents discussed above, itis contemplated that structurally similar compounds may be formulated tomimic the key portions of an immunoreactive peptide disclosed herein.Such compounds, which may be termed peptidomimetics, may be used in thesame manner as immunoreactive peptides disclosed herein and, hence, alsoare functional equivalents. Methods for generating specific structuresare disclosed, e.g., in Mizuno et al., 2017, as well as in U.S. Pat.Nos. 5,446,128; 5,710,245; 5,840,833; 5,859,184; 5,440,013; 5,618,914;and 5,670,155.

V. METHODS OF DETECTING EHRLICHIA INFECTION

Ehrlichiosis in humans generally refers to infections caused by obligateintracellular bacteria in the family Anaplasmataceae, chiefly in thegenera Ehrlichia and Anaplasma. The majority of cases of humanehrlichiosis (HE) are caused by 3 distinct species: Ehrlichiachaffeensis, chief among them (Dumler et al., 2007). Ehrlichiainfections in animals are also referred to as Ehrlichiosis, along with avariety of diseases caused by a diverse group of pathogens from genusesEhrlichia, Anaplasma, Neorickettsia, and Cowdria (Dumler et al., 2007).Ehrlichia infections are sustained mostly in monocytes or granulocytes,and studies have demonstrated that antibodies play an essential role inthe immune response to Ehrlichia infection (Feng and Walker, 2004;Winslow et al., 2003; Winslow et al., 2000; Yager et al., 2005).

Accordingly, select embodiments of the present disclosure providemethods of detecting antibodies that specifically bind an Ehrlichiaorganism in a sample. Such a method may involve contacting an isolatedehrlichial immunoreactive polypeptide comprising at least two peptidesof Table 1 or a polypeptide of Table 2, with the test sample, underconditions that allow peptide-antibody complexes to form, and detectingthe peptide-antibody complexes. In these embodiments, the detection ofthe peptide-antibody complexes is an indication that antibodies specificfor an Ehrlichia organism are present in the test sample, and theabsence of the peptide-antibody complexes is an indication thatantibodies specific an Ehrlichia organism are not present in the testsample.

In multiple embodiments, the detection of an immunoreactive polypeptidedisclosed herein bound to an Ehrlichia specific antibody (i.e., apeptide-antibody complex) may be accomplished using an enzyme-linkedimmunoassay (e.g., a sandwich ELISA, or a competitive ELISA), aradioimmunoassay, an immunoprecipitation, a fluorescence immunoassay, achemiluminescent assay, an immunoblot assay, a lateral flow assay, aflow cytometry assay, a mass spectrometry assay, latex agglutination, anindirect hemagglutination assay (IHA), complement fixation, aninhibition assay, an avidity assay, a dipstick test, or aparticulate-based assay. In some preferred embodiments, peptide-antibodycomplexes described herein are detected using an enzyme-linkedimmunoassay, a lateral flow assay, or a particle-based assay.

As used herein, a “sample” is any sample that comprises or is suspectedto comprise antibodies. Preferably, the sample is whole blood, sputum,serum, plasma, saliva, cerebrospinal fluid or urine. In someembodiments, the sample is a blood, serum or plasma sample obtained froma subject or patient.

Ehrlichiosis caused by an E. chaffeensis infection in humans presentswith flu-like symptoms of fever, chills, headache, and muscle aches. Inmore severe cases, nausea, loss of appetite, weight loss, abdominalpain, cough, diarrhea and change in mental status may also be observed.Ehrlichiosis in humans is potentially fatal.

In dogs, ehrlichiosis is most often caused by either E. chaffeensis orE. canis bacteria, and progresses in three phases: an acute phase, asubclinical phase, and a chronic phase. The acute phase normally extendsweeks after infection and features symptoms similar to those of humanehrlichiosis, such as fever, lethargy, loss of appetite, shortness ofbreath, joint pain and stiffness, and may also include more severesymptoms such as anemia, depression, bruising, and enlarged lymph nodes,liver, and spleen. The subclinical phase can persist for years and mostoften presents no symptoms, although antibodies to Ehrlichia antigensmay be detectable. The chronic phase of Ehrlichia infection generallyfeatures recurring symptoms of weight loss, anemia, neurologicaldysfunction, bleeding, ocular inflammation, leg edema, and fever, andpresents a blood profile which often leads to a misdiagnosis ofleukemia. An Ehrlichia infection that progresses to the chronic stage ofdisease is often fatal.

The nonspecific symptoms of an Ehrlichia infection and their resemblanceto mild and severe influenza symptoms makes diagnosis of Ehrlichiosisdifficult in humans and dogs. Diagnosis can be further hampered bycurrent laboratory testing procedures for Ehrlichia infection which arenot point-of-care tests, i.e., the tests are not available in mosthospitals, clinics, and physician or veterinarian offices where apatient can receive treatment.

Accordingly, select embodiments of the present disclosure providemethods of identifying an Ehrlichia infection in a mammalian subject.Such a method may involve contacting a sample from the subject with anisolated immunoreactive polypeptide disclosed herein, e.g., comprisingat least two peptides of Table 1, or more preferably a polypeptide ofTable 2, under conditions that allow peptide-antibody complexes to form,and detecting the peptide-antibody complexes. In these embodiments, thedetection of the peptide-antibody complexes is an indication that thesubject has an Ehrlichia infection. The Ehrlichia organism may be an E.chaffeensis organism or an E. canis organism. In some embodiments, thesubject is a human or a dog. As with other methods disclosed herein, thedetection step may be accomplished using any appropriate type of assayknown in the art, and may be preferrably accomplished using a lateralflow assay or an ELISA.

The terms “subject” and “patient” are used interchangeably herein, andmay refer to a mammal, especially a human or a dog. In certainembodiments, a “subject” or “patient” refers to a mammalian Ehrlichiahost (i.e., animal infected with an Ehrlichia organism). An Ehrlichiahost may be, for example, human or non-human primate, bovine, canine,caprine, cavine, corvine, epine, equine, feline, hircine, lapine,leporine, lupine, murine, ovine, porcine, racine, vulpine, and the like,including livestock, zoological specimens, exotics, as well as companionanimals, pets, and any animal under the care of a veterinarypractitioner. A subject may be or may not be infected with an Ehrlichiaorganism, and a subject may be a mammal suspected of being infected withan Ehrlichia organism.

Without wishing to be bound by theory, the ehrlichial immunoreactivepolypeptides disclosed herein each comprise at least a part of a majorEhrlichia epitope that accounts for a species-specific immunogenicity inhumans and animals. The term “epitope” is used herein to indicate thatportion of an immunogenic substance that is specifically identified,recognized, and bound by, an antibody or cell-surface receptor of a hostimmune system that has mounted an immune response to the immunogenicsubstance as determined by any method known in the art. (see, forexample, Geysen et al., 1984). Thus, an epitope that is“species-specific” is an epitope that can be used to differentiate onespecies of the Ehrlichia genus from another Ehrlichia species.

Particular embodiments relate to determining whether a subject has beenimmunized against Ehrlichia or is actively infected with an Ehrlichiaorganism. In these embodiments, the method comprises contacting a samplefrom the subject with at least one isolated immunoreactive peptides ofTable 1, or more preferably a polypeptide of Table 2, that is not acomponent of an Ehrlichia vaccine, and detecting whether an antibody inthe sample specifically binds to the isolated ehrlichial immunoreactivepolypeptide. According to the method, if an antibody in the samplespecifically binds to the isolated ehrlichial immunoreactivepolypeptide, then the subject has an active Ehrlichia infection, and ifan antibody does not specifically bind to the isolated ehrlichialimmunoreactive peptide, then the subject is either previously immunizedwith an Ehrlichia vaccine or is not infected with an Ehrlichia organism.An Ehrlichia organism may be an E. chaffeensis organism or an E. canisorganism.

An ehrlichial immunoreactive polypeptide (e.g., comprising at least twopeptides of Table 1 or a polypeptide of Table 2) may be used to bind anEhrlichia-specific or E. chaffeensis-specific antibody using a varietyof methods or kits. The specific binding between an antibody and animmunoreactive peptide of the present disclosure may therefore beassessed by any appropriate method known in the art including, but notlimited to, an enzyme-linked immunosorbent assay (ELISA), a sandwichELISA, a competitive ELISA, immunoblotting, immunoprecipitation,radioimmunoassay (RIA), immunostaining, latex agglutination, indirecthemagglutination assay (IHA), complement fixation, indirectimmnunofluorescent assay (FA), nephelometry, flow cytometry assay,chemiluminescence assay, lateral flow immunoassay, u-capture assay, massspectrometry assay, particle-based assay, inhibition assay and avidityassay. Exemplary methods of detecting the binding of anEhrlichia-specific antibody to an ehrlichial immunoreactive polypeptideas disclosed herein may include, for example, an ELISA performed in amicroplate, a lateral flow test performed using a dipstick or lateralflow device, or a particulate-based suspension array assay performedusing the Bio-Plex® system (Bio-Rad Laboratories, Hercules, Calif.,USA).

A. ELISA

In certain embodiments, the detection of a peptide-antibody complexdescribed herein is accomplished using an enzyme linked immunosorbentassay (ELISA). This assay may be performed by first contacting anehrlichial immunoreactive polypeptide (e.g., comprising at least twopeptides of Table 1 or a polypeptide of Table 2) that has beenimmobilized on a solid support, commonly the well of a microtiter plate,with the sample, such that antibodies specific for the peptide withinthe sample are allowed to bind to the immobilized peptide. Unboundsample is then removed from the immobilized peptide and a detectionreagent capable of binding to the immobilized antibody-polypeptidecomplex is added. The amount of detection reagent that remains bound tothe solid support is then determined using a method appropriate for thespecific detection reagent.

In some embodiments, the detection reagent contains a binding agent(such as, for example, Protein A, Protein G, immunoglobulin, lectin orfree antigen) conjugated or covalently attached to a reporter group orlabel. Exemplary reporter groups or labels include enzymes (such ashorseradish peroxidase), substrates, cofactors, inhibitors, dyes,radionuclides, luminescent groups, fluorescent groups and biotin. Theconjugation of binding agent to reporter group or label may be achievedusing standard methods known to those of ordinary skill in the art.Common binding agents may also be purchased conjugated to a variety ofreporter groups from many commercial sources (e.g., Zymed Laboratories,San Francisco, Calif.; and Pierce, Rockford, Ill.).

In an aspect of the present disclosure, the presence or absence ofEhrlichia specific antibodies may be determined in the sample bycomparing the level of a signal detected from a reporter group or labelin the sample with the level of a signal that corresponds to a controlsample or predetermined cut-off value. In certain embodiments, thecut-off value may be the average mean signal obtained when theimmobilized ehrlichial immunoreactive peptide is incubated with samplesfrom an uninfected subject. The cut-off value may be determined using astatistical method or computer program.

B. Lateral Flow Tests

Lateral flow tests may also be referred to as immunochromatographicstrip (ICS) tests or simply strip-tests. In general, a lateral flow testis a form of assay in which the test sample flows laterally along asolid substrate via capillary action, or alternatively, under fluidiccontrol. Such tests are often inexpensive, require a very small amount(e.g., one drop) of sample, and can typically be performed reproduciblywith minimal training. The economical simplicity and robustness of manylateral flow assay formats makes these types of tests ideal foridentifying an Ehrlichia (e.g., E. chaffeensis or E. canis) infection atthe point of care, which can be particularly important when the subjectis, for example, a human or dog exhibiting detectable antibodies duringthe treatable acute phase of infection.

Exemplary lateral flow device formats include, but are not limited to, adipstick, a card, a chip, a microslide, and a cassette, and it is widelydemonstrated in the art that the choice of format is largely dependentupon the features of a particular assay. Accordingly, lateral flowdevices are now ubiquitous in human and veterinarian medicine and quitevaried, providing many options to the ordinarily skilled artisan fordetecting a peptide-antibody complex in a sample using a lateral flowassay (See any of U.S. Pat. Nos. 7,344,893, 7,371,582, 6,136,610, andU.S. Patent Applications, 2005/0250141 and 2005/0047972, or Koczula etal. (2016) each incorporated herein by reference.) By way of anonlimiting example, a sample from a subject suspected of having anEhrlichia infection is applied to a lateral flow device comprising atleast a sample zone and a binding zone. The sample may be a serumsample, and may be drawn laterally from the sample zone to the bindingzone which comprises an ehrlichial immunoreactive polypeptide disclosedherein (e.g., comprising at least two peptides of Table 1 or apolypeptide of Table 2) immobilized to a surface of the lateral flowdevice. In this example, the binding of the immobilized ehrlichialimmunoreactive polypeptide on the lateral flow device is an indicationthat Ehrlichia specific antibodies are present in the sample from thesubject, indicating an Ehrlichia infection in the subject, such as an E.chaffeensis infection in the subject.

In related embodiments, an ELISA assay as described above may beperformed in a rapid flow-through, lateral flow, or strip test format,wherein the antigen is immobilized on a membrane, such as anitrocellulose membrane. In this flow-through test, Ehrlichia antibodieswithin the sample bind to the immobilized ehrlichial immunoreactivepeptide as the sample passes through the membrane. A detection reagent,such as protein A labeled with gold, a fluorophore, or a chromophore,binds to the peptide-antibody complex as the solution containing thedetection reagent flows through the membrane. Peptide-antibody complexesbound to detection reagent may then be detected, as appropriate for thedetection reagent used (e.g., based on the presence or absence of avisibly detectable color or fluorescent label, a nanoparticle, aluminescent rare earth nanoparticle, a luminous nanoparticle, astrontium aluminate nanoparticle (e.g., see Paterson et al., 2014; andWang et al., 2017, etc.).

In an aspect, a flow-through format ELISA may be performed in which oneend of the membrane to which an ehrlichial immunoreactive peptide (e.g.,comprising at least two peptides of Table 1 or a polypeptide of Table 2)is immobilized may be immersed in a solution containing the sample, orthe sample may be added to an area (i.e., a sample zone) at one end ofthe membrane. The sample migrates along the membrane through a region(i.e., a labeling zone) comprising the detection reagent, and flows tothe area (i.e., a binding zone) comprising the immobilized ehrlichialimmunoreactive peptide. An accumulation of detection reagent at thebinding zone indicates the presence of Ehrlichia specific antibodies inthe sample.

Typically, a flow-through ELISA may feature a detection reagent appliedto a test strip in a pattern, such as a line, that can be read visually.As with other lateral flow tests, the absence of such a patterntypically indicates a negative result. It is within the ability of anordinarily skilled artisan to select an amount of the ehrlichialimmunoreactive polypeptide for immobilization on the membrane that cangenerate a visually discernible pattern when the biological samplecontains a level of antibodies that would be sufficient to generate apositive signal in a standard format ELISA. Preferably, the amount ofpeptide immobilized on the membrane ranges from about 25 ng to about 1mg.

C. Particulate-Based Assays

In general, particle-based assays use a capture-binding partner, such asan antibody or an antigen in the case of an immunoassay, coated on thesurface of particles, such as microbeads, crystals, chips, ornanoparticles. Particle-based assays may be effectively multi-plexed ormodified to assay numerous variables of interest by incorporatingfluorescently labeled particles or particles of different sizes in asingle assay, each coated or conjugated to one or more labeledcapture-binding partners. The use of sensitive detection andamplification technologies with particle-based assay platforms known inthe art has resulted in numerous flexible and sensitive assay systems tochoose from in performing a method described herein. For example, amultiplex particle-based assay such as the suspension array Bio-Plex®assay system available from Bio-Rad Laboratories, Inc. (Hercules,Calif.) and Luminex, Inc. (Austin, Tex.) may be useful in identifyingEhrlichia antibodies in a sample.

In an aspect, the present disclosure contemplates the immobilization ofan isolated ehrlichial immunoreactive polypeptide (e.g., comprising atleast two peptides of Table 1 or a polypeptide of Table 2) on a surfaceof a particle for use in a particle-based immunoassay. As describedherein, methods of peptide immobilization onto support surfaces is wellknown in the art. In a preferred embodiment, a labeled herimmunoreactive polypeptide disclosed herein is immobilized onto asurface of a particle and the peptide-particle complex is employed in anELISA or in a flow cytometry assay according to established protocols.

VI. EHRLICHIA VACCINE AND IMMUNOGENIC COMPOSITIONS

Previous work has shown that Ehrlichial proteins that induce antibodyresponses can provide protective immune responses; thus, in someembodiments an ehrlichial protein provided herein (e.g., a polypeptideof Formula I or a polypeptide of Table 2) may be included in apharmaceutical composition such as a vaccine composition foradministration to a mammalian or human subject. For example, protectionagainst E. chaffeensis infection has been demonstrated withepitope-specific antibodies directed at OMP and TRPs in in vitro modelsand in animal models (Kuriakose et al., 2012; Li et al., 2002; Li etal., 2001), demonstrating that ehrlichial proteins that elicit strongantibody responses to linear epitopes are protective.

In select embodiments, it is contemplated that an ehrlichialimmunoreactive polypeptide of Formula I or a polypeptide of Table 2 maybe comprised in a vaccine composition and administered to a subject(e.g., a human or dog) to induce a protective immune response in thesubject that may substantially prevent or ameliorate infection in thesubject by an Ehrlichia organism such as Ehrlichia chaffeensis orEhrlichia canis. A vaccine composition for pharmaceutical use in asubject may comprise an immunoreactive polypeptide of Formula I or apolypeptide of Table 2 and a pharmaceutically acceptable carrier.

The phrases “pharmaceutical,” “pharmaceutically acceptable,” or“pharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, surfactants,antioxidants, preservatives (e.g., antibacterial agents, antifungalagents), isotonic agents, absorption delaying agents, salts,preservatives, drugs, drug stabilizers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, such like materials and combinations thereof, as would be known toone of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1289-1329,1990, incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the vaccine compositions of the present disclosure is contemplated.

As used herein, a “protective immune response” refers to a response bythe immune system of a mammalian host to an Ehrlichia antigen whichresults in increased recognition of the antigen and antibody productionby the immune system of the mammalian host upon subsequent exposure toan Ehrlichia pathogen. A protective immune response may substantiallyreduce or prevent symptoms as a result of a subsequent exposure toEhrlichia chaffeensis or Ehrlichia canis.

In some embodiments, a vaccine composition of the present disclosure maycomprise an immunoreactive polypeptide (e.g., having a sequence that hasat least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to a polypeptide of Formula I or a polypeptide ofTable 2). In some embodiments, a vaccine composition comprising theimmunoreactive polypeptide may be used to induce a protective immuneresponse against Ehrlichia chaffeensis (e.g., in a human or dogsubject).

A person having ordinary skill in the medical arts will appreciate thatthe actual dosage amount of a vaccine composition administered to ananimal or human patient can be determined by physical and physiologicalfactors such as body weight, severity of condition, the type of diseasebeing treated, previous or concurrent therapeutic interventions,idiopathy of the patient and on the route of administration. Thepractitioner responsible for administration will, in any event,determine the concentration of active ingredient(s) in a composition andappropriate dose(s) for the individual subject.

In certain embodiments, vaccine compositions may comprise, for example,at least about 0.1% of an ehrlichial immunoreactive polypeptidecomprising or consisting of a polypeptide of Formula I or Table 2. Inother embodiments, the active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. As with many vaccinecompositions, frequency of administration, as well as dosage, will varyamong members of a population of animals or humans in ways that arepredictable by one skilled in the art of immunology. By way ofnonlimiting example, the pharmaceutical compositions and vaccines may beadministered by injection (e.g., intracutaneous, intramuscular,intravenous or subcutaneous), intranasally (e.g., by aspiration) ororally. Between 1 and 3 doses may be administered over a 1-36 weekperiod. In some embodiments, 3 doses are administered, at intervals of3-4 months, and booster vaccinations may be given periodicallythereafter.

In some embodiments, a “suitable dose” is an amount of an immunoreactivepolypeptide that, when administered as described above, is capable ofraising an immune response in an immunized patient sufficient to protectthe subject from an Ehrlichia infection in subsequent exposures toEhrlichia organisms. In general, the amount of peptide present in asuitable dose (or produced in situ by the nucleic acid in a dose) mayrange from about 1 pg to about 500 mg per kg of host, typically fromabout 10 pg to about 10 mg, preferably from about 100 pg to about 1 mgand more preferably from about 100 pg to about 100 microgram.

A vaccine composition of the present disclosure may comprise differenttypes of carriers depending on whether it is to be administered insolid, liquid or aerosol form, and whether it needs to be sterile forsuch routes of administration as injection. A vaccine compositiondisclosed herein can be administered intramuscularly, intradermally,subcutaneously, intravenously, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostaticaly,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrarectally, topically, intratumorally,intramuscularly, intraperitoneally, subconjunctivally, intravesicularly,mucosally, intrapericardially, locally, orally, intranasally, or byinhalation, injection, infusion, continuous infusion, lavage, orlocalized perfusion. A vaccine composition may also be administered to asubject via a catheter, in cremes, in lipid compositions, by ballisticparticulate delivery, or by other method or any combination of theforgoing as would be known to one of ordinary skill in the art (see, forexample, Remington: The Science and Practice of Pharmacy, 21^(st) Ed.Lippincott Williams and Wilkins, 2005, incorporated herein byreference).

While any suitable carrier known to those of ordinary skill in the artmay be employed in the vaccine compositions of this invention, the typeof carrier will vary depending on the mode of administration. Forparenteral administration, such as subcutaneous injection, the carrierpreferably comprises water, saline, alcohol, a fat, a wax or a buffer.For oral administration, any of the above carriers or a solid carrier,such as mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, glucose, sucrose, and magnesiumcarbonate, may be employed. Biodegradable microspheres (e.g., polylacticgalactide) may also be employed as carriers for the pharmaceuticalcompositions of this invention. Suitable biodegradable microspheres aredisclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109.

Of particular interest, in some embodiments, is a vaccine compositionthat may be administered by microstructured transdermal or ballisticparticulate delivery. Microstructures as carriers for vaccineformulation are a desirable configuration for vaccine applications andare widely known in the art (e.g., U.S. Pat. Nos. 5,797,898, 5,770,219and 5,783,208, and U.S. Patent Application 2005/0065463). Such a vaccinecomposition formulated for ballistic particulate delivery may comprisean isolated immunoreactive polypeptide of Table 1, 2, or 3 immobilizedon a surface of a support substrate. In these embodiments, a supportsubstrate can include, but is not limited to, a microcapsule, amicroparticle, a microsphere, a nanocapsule, a nanoparticle, ananosphere, or a combination thereof.

Microstructures or ballistic particles that serve as a support substratefor an ehrlichial immunoreactive polypeptide disclosed herein may becomprised of biodegradable material and non-biodegradable material, andsuch support substrates may be comprised of synthetic polymers, silica,lipids, carbohydrates, proteins, lectins, ionic agents, crosslinkers,and other microstructure components available in the art. Protocols andreagents for the immobilization of a peptide of the invention to asupport substrate composed of such materials are widely availablecommercially and in the art.

In other embodiments, a vaccine composition comprises an immobilized orencapsulated immunoreactive polypeptide of Formula I or a polypeptide ofTable 2 and a support substrate. In these embodiments, a supportsubstrate can include, but is not limited to, a lipid microsphere, alipid nanoparticle, an ethosome, a liposome, a niosome, a phospholipid,a sphingosome, a surfactant, a transferosome, an emulsion, or acombination thereof. The formation and use of liposomes and other lipidnano- and microcarrier formulations is generally known to those ofordinary skill in the art, and the use of liposomes, microparticles,nanocapsules and the like have gained widespread use in delivery oftherapeutics (e.g., U.S. Pat. No. 5,741,516, specifically incorporatedherein in its entirety by reference). Numerous methods of liposome andliposome-like preparations as potential drug carriers, includingencapsulation of peptides, have been reviewed (U.S. Pat. Nos. 5,567,434;5,552,157; 5,565,213; 5,738,868 and 5,795,587, each of which isspecifically incorporated in its entirety by reference).

In addition to the methods of delivery described herein, a number ofalternative techniques are also contemplated for administering thedisclosed vaccine compositions. By way of nonlimiting example, a vaccinecomposition may be administered by sonophoresis (i.e., ultrasound) whichhas been used and described in U.S. Pat. No. 5,656,016 for enhancing therate and efficacy of drug permeation into and through the circulatorysystem; intraosseous injection (U.S. Pat. No. 5,779,708), orfeedback-controlled delivery (U.S. Pat. No. 5,697,899), and each of thepatents in this paragraph is specifically incorporated herein in itsentirety by reference.

Any of a variety of adjuvants may be employed in the vaccines of thisinvention to nonspecifically enhance the immune response. Most adjuvantscontain a substance designed to protect the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and a nonspecificstimulator of immune responses, such as lipid A, Bortadella pertussis orMycobacterium tuberculosis. Suitable adjuvants are commerciallyavailable as, for example, Freund's Incomplete Adjuvant and Freund'sComplete Adjuvant (Difco Laboratories, Detroit, Mich.) and MerckAdjuvant 65 (Merck and Company, Inc., Rahway, N.J.). Other suitableadjuvants include alum, biodegradable microspheres, monophosphoryl lipidA and quil A.

A polypeptide may be formulated into a composition in a neutral or saltform. Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids such as acetic, oxalic, tartaric, mandelic,and the like. Salts formed with the free carboxyl groups can also bederived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, histidine, procaine and the like.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

Sterile injectable solutions are prepared by incorporating the activepeptides in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle that contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less than 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

A. Ehrlichia Bacterin

In some embodiments, an immunogenic or vaccine composition as disclosedherein comprises an Ehrlichia bacterin, such as an E. canis bacterin oran E. chaffeensis bacterin. E Canis bacterin may be prepared byheat-inactivating or chemically-inactivating the Ehrlichia bacteria.

A variety of methods may be used to generate an E. chaffeensis or E.canis bacterin. For example, the bacteria may be inactivated by heat orpsoralen in the presence of ultraviolet light to produce the bacterin.The effective immunizing amount of the inactivated Ehrlichia bacterincan vary depending upon the chosen strain or strains. It is anticipatedthat any amount of an Ehrlichia bacterin, alone or in combination with(i) chimeric polypeptide as disclosed herein (e.g., a polypeptide ofFormula I or of Table 2), and/or (ii) adjuvant(s), sufficient to evoke aprotective immune response may be used in various embodiments (e.g., toinduce a protective immune response in a subject). In some embodiments,a dosage unit comprising at least about 1×10⁴ TCID₅₀ inactivated E.chaffeensis and/or E. canis bacterin can be used. Additional methodsthat may be used to generate an Ehrlichia bacterin include, but are notlimited to, treatment of an E. chaffeensis or E. canis with heat,formaldehyde, formalin, bi-ethylene amine, radiation, and/orbeta-propiolactone treatment. It is anticipated that the bacterin may beinactivated by any suitable method available. Additional methods thatmay be used to generate an Ehrlichia or E. canis bacterin include thosedescribed, e.g., in WO2005087803, EP2433646, Vega et al., 2007; or Stuenet al., 2015.

In some embodiments, the Ehrlichia bacterin comprises inactivated crudeantigen based on inactivated E. chaffeensis and/or E. canis bacteria.For example, in some embodiments, frozen buffy coat (e.g., 10 ml frozenbuffy coat) containing E. chaffeensis or E. Canis may be obtained, andthe material was inactivated using 0.3% formaldehyde for 48 h at roomtemperature. Thereafter, the material can tested for lack of infectivityby in vitro methods or by using an in vivo animal model. Methods forinactivating bacteria using formaldehyde are further described inTollersrud et al., 2001. The resulting Ehrlichia bacterin can beincluded with (i) 1, 2, 3, or more chimeric immunogenic proteins orpeptides as described herein (e.g., as described in Table 2) and/or (ii)an adjuvant, to form an immunogenic or vaccine composition. For example,the inactivated E. canis bacterin may be prepared as a suspension andthen included in an emulsion adjuvant, e.g., as described below.

B. Adjuvants

In some aspects, an immunogenic composition comprising one or morechimeric polypeptide as disclosed herein (e.g., a polypeptide of FormulaI or of Table 2) also contains an adjuvant. In some embodiments, thecomposition is a pharmaceutical preparation or a vaccine composition. Avariety of adjuvants are known that can be included. For example,adjuvants such as MF59, AS01, AS02, AS03, AS04, Virosomes, CAF01, CAF04,CAF05, Montanide ISA™ 720, or Montanide ISA™ 51 (e.g., Bonam et al.,2017) can be used in some embodiments.

In some embodiments, the immunogenic or vaccine composition includes anadjuvant comprising a triterpenoid, sterol, immunomodulator, polymer,and/or Th2 stimulator. For example, in some embodiments the adjuvantcomprises DEAE Dextran, an immunostimulatory oligonucleotide, and oil(e.g., a light mineral oil), wherein the immunostimulatoryoligonucleotide is a CpG containing ODN, and wherein the adjuvantformulation is a water-in-oil (W/O) emulsion. The vaccine adjuvant mayoptionally comprise an Ehrlichia bacterin (such as a heat-inactivated E.Canis or E. chaffeensis) and/or a chimeric peptide as disclosed herein(e.g., of Formula I or Table 2). In some embodiments, the immunogenic orvaccine composition includes an antigen component and an adjuvantformulation comprising a saponin (e.g., present in an amount of about 1μg to about 5,000 μg per dose), a sterol (e.g., present in an amount ofabout 1 μg to about 5,000 μg per dose), a quaternary ammonium compound(e.g., present in an amount of about 1 μg to about 5,000 .mu.g perdose), a polymer (e.g., present in an amount of about 0.0001% v/v toabout 75% v/v.), and an ORN/ODN; the saponin may be Quil A or a purifiedfaction thereof, the sterol may be cholesterol, the quaternary ammoniumcompound may be dimethyl dioctadecyl ammonium bromide (DDA), the polymermay be polyacrylic acid, and the ORN/ODN may be a CpG. The adjuvant maycomprise a glycolipid, suchN-(2-deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyldodecanamideacetate. The adjuvant may comprise an immunostimulatory oligonucleotide,a polyacrylic acid polymer and at least two of the following: (a)dimethyl dioctadecyl ammonium bromide (DDA); (b) a sterol; and/or (c)N-(2-deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyldodecanamideacetate. For example, the vaccine composition may comprise an adjuvantas described, e.g., in U.S. Pat. Nos. 10,238,736, 8,580,280, or USPublication 2019/0008953.

In some embodiments, immunogenic or vaccine composition includes anantigen component and an adjuvant formulation comprising a triterpenoidsaponin, a sterol, a quaternary ammonium compound, and a polyacrylicacid polymer, wherein the antigen component comprises or consists of aEhrlichia bacterin (such as a heat-inactivated E. canis) and/or achimeric polypeptide as disclosed herein (e.g., a polypeptide of FormulaI or of Table 2). In some embodiments, the saponin is present in anamount of about 1 mg to about 5,000 mg per dose, the sterol is presentin an amount of about 1 mg to about 5,000 mg per dose, the quaternaryammonium compound is present in an amount of about 1 mg to about 5,000mg per dose, and the polyacrylic acid polymer is present in an amount ofabout 0.0001% v/v to about 75% v/v. For example, the vaccine compositionmay comprise an adjuvant as described, e.g., in U.S. Pat. No. 9,662,385.

In some aspects, an immunogenic or vaccine composition as disclosedherein comprises an oil-based adjuvant comprising an Ehrlichia bacterin(such as a heat-inactivated E. canis or E. chaffensis) and/or one ormore chimeric polypeptide as disclosed herein (e.g., a polypeptide ofFormula I or of Table 2). For example, the adjuvant formulation maycomprise an oily phase and an aqueous phase, a polycationic carrier(e.g., DEAE dextran), and a CpG containing immunostimulatoryoligonucleotide, wherein the vaccine is a water-in-oil emulsion. Theadjuvant may optionally further comprise an aluminum hydroxide gel. Insome embodiments, the CpG containing immunostimulatory oligonucleotideis present in the amount of about 50 to about 400 μg per dose and DEAEDextran is present in the amount of about 10 to about 300 mg per dose.The adjuvant formulation may comprise an immunostimulatingoligonucleotide, polycationic carrier, sterol, saponin, quaternaryamine, TLR-3 agonist, glycolipid, and/or MPL-A (or an analog thereof) inan oil emulsion. For example, the vaccine composition may comprise anadjuvant as described, e.g., in U.S. Pat. No. 10,117,921 or US2019/0038737.

In some embodiments, the immunogenic composition is an emulsioncomprising (i) an Ehrlichia bacterin (such as a heat-inactivated E.canis or E. chaffeensis), and/or (ii) one or more chimeric polypeptideas disclosed herein (e.g., a polypeptide of Formula I or of Table 2).For example, the emulsion composition may comprise an adjuvant, such asacrylic polymer and/or dimethyl dioctadecyl ammonium bromide (DDA), inthe aqueous phase. The emulsion can be prepared, in some embodiments, bymixing an aqueous phase containing the antigen (e.g., an E. canisbacterin such as a heat-inactivated E. canis, and/or one or morechimeric polypeptide (e.g., a polypeptide of Formula I or of Table 2))and adjuvant with an oil phase in the presence of an emulsifier. In someembodiments, the adjuvant component comprises an oil-in-water emulsion,wherein the aqueous phase of the oil-in-water emulsion comprisesdimethyl dioctadecyl ammonium bromide (DDA) and/or an alkyl-polyacrylicacid (alkyl-PAA). In some embodiments, the oil in the oil-in-wateremulsion is mineral oil, a terpene oil, soybean oil, olive oil, or apropylene glycol derivative. The adjuvant may further comprise theadjuvant component further comprises CpG DNA, a lipopolysaccharide,and/or monophosphoryl lipid A. The vaccine may further comprise one ormore emulsifiers. For example, the vaccine composition may comprise anadjuvant as described, e.g., in U.S. Pat. No. 9,545,439 or 8,980,288.

The adjuvant may be a liposome or emulsion formulation. The liposomesmay be unilamellar, multilamellar, or multivesicular. In someembodiments, the an immunogenic or vaccine composition comprises a lipidor lipid-containing adjuvant. In some embodiments, the liposomes arecationic liposomes. In various embodiments, adjuvants such as MF59(e.g., Calabro et al. (2013) Vaccine 31: 3363-3369), AS01(Didierlaurent, et al. (2014) J. Immunol. 193, 1920-1930), AS02 (Garconand Van Mechelen (2011) Expert Rev. Vaccines 10, 471-486), AS03 (Morel,S. et al. (2011) Vaccine 29, 2461-2473), AS04 (Didierlaurent, et al.(2009) J. Immunol. 183: 6186-6197.), Virosomes (Künzi, et al. (2009)Vaccine 27, 3561-3567), CAF01 (Tandrup Schmidt, et al. (2016)Pharmaceutics 8, 7.), CAF04 (Billeskov, et al. (2016) PLoS One 11,e0161217), CAF05 (Billeskov, et al. (2016) PLoS One 11, e0161217),Montanide ISA™ 720 (Aucouturier, et al. (2002) Expert Rev. Vaccines 1,111-118), or Montanide ISA™ 51 (Aucouturier, et al. (2002) Expert Rev.Vaccines 1, 111-118) can be used. Table 3 provides a listing of exampleadjuvant containing formulations that can be used in variousembodiments.

TABLE 3 Example adjuvant containing formulations Adjuvant CompositionMF59 Squalene, Span 85, Tween 80, and citrate buffer AS01 Liposomescontaining 3-O-desacyl-4′- monophosphoryl lipid A (MPLA) and QS21 AS02Oil-in-water (O/W) emulsion containing MPLA and the saponin QS21 AS03α-tocopherol, squalene, polysorbate 80, and PBS AS04 Contains MPLAadsorbed onto a particulate form of aluminum salt Virosomes Containinactivated virus CAF01 Cationic liposomal vehicle containing dimethyldioctadecyl-ammonium (DDA) with a glycolipid immunostimulator (TDB)CAF04 Cationic liposomal vehicle containing DDA with monomycoloylglycerol analog (MMG) CAF05 Cationic liposomal vehicle containing DDAwith the immunostimulators TDB and poly(I:C) Montanide Water-in-oil(W/O) emulsion containing non- ISA ™ 720 mineral oil with mannidemono-oleate family emulsifier Montanide W/O emulsion containing mineraloil with ISA ™ 51 mannide mono-oleate family emulsifier Acrylic polymer/Oil-in-water emulsion comprises dimethyl DDA emulsions dioctadecylammonium bromide (DDA) and/or an alkyl-polyacrylic acid (alkyl-PAA);e.g., see U.S. Pat. No. 9,545,439 or U.S. Pat. No. 8,980,288. CpG/DEAEEmulsions comprising a polycationic carrier emulsions (e.g., DEAEdextran) and a CpG containing immunostimulatory oligonucleotide; e.g.,see U.S. Pat. No. 10,117,921 or US 2019/0038737. Saponin/cholesterol/Saponin (e.g., Quil A), cholesterol, DDA, a DDA adjuvants polyacrylicacid; e.g., a triterpenoid saponin, a sterol, a quaternary ammoniumcompound, and a polyacrylic acid polymer; e.g., see U.S. Pat. No.9,662,385. Polyacrylic acid Water-in-oil (W/O) emulsions, DEAE Dextran,polymer emulsions immunostimulatory oligonucleotide (e.g., a CpGcontaining ODN), a sterol, N-(2-deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N- octadecyldodecanamide acetate, and/ora polyacrylic acid polymer; e.g., see U.S. Pat. No. 10,238,736, U.S.Pat. No. 8,580,280, or US Publication 2019/0008953.

VII. EHRLICHIA DETECTION AND VACCINATION KITS

Various embodiments of the present disclosure are concerned with kitsfor the detection of antibodies in a sample that specifically bind anEhrlichia organism, such as E. chaffeensis or E canis. The kits may thusbe used for the diagnosis or identification of an Ehrlichia infection ina subject. In other embodiments, the invention provides kits fordetermining whether a subject has been immunized against Ehrlichia or isactively infected with an Ehrlichia organism. In still otherembodiments, kits are provided for vaccination of a subject against E.chaffeensis infection, and in some embodiments it is anticipated thatthe composition may be used to provide a protective immune responseagainst an E. canis infection.

In select embodiments, a kit of the present disclosure may be used toperform a method disclosed herein. For example, a kit may be suitablefor detecting Ehrlichia antibodies in a sample, for identifying anEhrlichia infection individual, for determining whether a subject hasbeen immunized against Ehrlichia or is actively infected with anEhrlichia organism, or for vaccinating a subject against an Ehrlichiaorganism. In these embodiments, one or more immunoreactive peptide(e.g., comprising a polypeptide of Formula I or a polypeptide of Table2, or a polypeptide having at least about 95% or more sequence identitywith a polypeptide of Formula I or a polypeptide of Table 2) may becomprised in the kit. The ehrlichial immunoreactive polypeptide in thekit may be detectably labeled or immobilized on a surface of a supportsubstrate also comprised in the kit. The immunoreactive polypeptide(s)may, for example, be provided in the kit in a suitable form, such assterile, lyophilized, or both.

The support substrate comprised in a kit of the invention may beselected based on the method to be performed. By way of nonlimitingexample, a support substrate may be a multi-well plate or microplate, amembrane, a filter, a paper, an emulsion, a bead, a microbead, amicrosphere, a nanobead, a nanosphere, a nanoparticle, an ethosome, aliposome, a niosome, a transferosome, a dipstick, a card, a celluloidstrip, a glass slide, a microslide, a biosensor, a lateral flowapparatus, a microchip, a comb, a silica particle, a magnetic particle,or a self-assembling monolayer.

As appropriate to the method being performed, a kit may further compriseone or more apparatuses for delivery of a composition to a subject orfor otherwise handling a composition of the invention. By way ofnonlimiting example, a kit may include an apparatus that is a syringe,an eye dropper, a ballistic particle applicator (e.g., applicatorsdisclosed in U.S. Pat. Nos. 5,797,898, 5,770,219 and 5,783,208, and U.S.Patent Application 2005/0065463), a scoopula, a microslide cover, a teststrip holder or cover, and such like.

A detection reagent for labeling a component of the kit may optionallybe comprised in a kit for performing a method of the present disclosure.In particular embodiments, the labeling or detection reagent is selectedfrom a group comprising reagents used commonly in the art and including,without limitation, radioactive elements, enzymes, molecules whichabsorb light in the UV range, and fluorophores such as fluorescein,rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. In otherembodiments, a kit is provided comprising one or more container meansand a BST protein agent already labeled with a detection reagentselected from a group comprising a radioactive element, an enzyme, amolecule which absorbs light in the UV range, and a fluorophore.

In particular embodiments, the present disclosure provides a kit fordetecting anti-Ehrlichia antibodies in a sample which may also be usedfor identification of an Ehrlichia infection in a subject, and/or fordetermining whether a subject has been immunized against Ehrlichia or isactively infected with an Ehrlichia organism. Such a kit may compriseone or more immunoreactive polypeptides (e.g., comprising a polypeptideof Formula I or a polypeptide of Table 2, or having at least about 95%or more sequence identity with a polypeptide comprising a polypeptide ofFormula I or a polypeptide of Table 2), and the peptides may bedetectably labeled and immobilized to one or more support substratescomprised in the kit.

In some embodiments, a kit comprises an immunoreactive polypeptidecomprising a polypeptide of Formula I or a polypeptide of Table 2 orhaving about 95% or more sequence identity with polypeptide comprising apolypeptide of Formula I or a polypeptide of Table 2. The peptides maybe immobilized to one or more separate lateral flow assay devices, suchas a nitrocellulose test strips. In these embodiments, each of the teststrips may further comprises a detection reagent, for example, achromophore-labeled protein A. Such a kit may further comprise one ormore containers for sample material, one or more diluents for sampledilution, and one or more control indicator strips for comparison.

When reagents and/or components comprising a kit are provided in alyophilized form (lyophilisate) or as a dry powder, the lyophilisate orpowder can be reconstituted by the addition of a suitable solvent. Inparticular embodiments, the solvent may be a sterile, pharmaceuticallyacceptable buffer and/or other diluent. It is envisioned that such asolvent may also be provided as part of a kit.

When the components of a kit are provided in one and/or more liquidsolutions, the liquid solution may be, by way of non-limiting example, asterile, aqueous solution. The compositions may also be formulated intoan administrative composition. In this case, the container means mayitself be a syringe, pipette, topical applicator or the like, from whichthe formulation may be applied to an affected area of the body, injectedinto a subject, and/or applied to or mixed with the other components ofthe kit.

IV. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Methods

Chimera Construction and Cloning

Sequences representing five Ehrlichia epitope chimeras were synthesizedcommercially (GenScript). The chimeric sequences were cloned into pET-14and the recombinant chimeric protein expressed in E. coli BL21 (DE) orBL21-AI (Invitrogen) (Table 1).

TABLE 1 Ehrlichia chimeras Expression Chimera Name MW pl Vector cell TagSolubility E. chaff TRP32/TRP120/A34 30.8 KD 3.79 pET-14b BL21-(DE3) HisSoluble E. canis TRP140/TRP36/TRP19 22.8 KD 3.94 pET-14b BL21-(DE3) HisSoluble E. canis TRP36/TRP140 17.8 KD 3.93 pET-14b BL21-Al His SolubleEhrlichia TRP32/TRP120/ 31.4 KD 4.13 pET-14b BL21-(DE3) His SolubleTRP36/TRP140/P28/HSP Ehrlichia TRP120/TRP140/ 27.9 KD 4.09 pET-14bBL21-Al His Soluble TRP36/P28

Chimera Expression and Purification

Ehrlichia chimera recombinant proteins were purified under nativeconditions using Roche cOmplete™ His-Tag Protein Purification Protocol.Briefly, E. coli cell pellets were resuspended in lysis buffer (50 mMTris-HCl, 150 mM NaCl, 2 mMDTT, 2 mM MgCl2, 5 mM EDTA and 5 mMImidazole) and sonicated on ice for 5 min, then lysates were cleared bycentrifugation for 1 h at 12,000×g at 4° C. His-tagged proteins werepurified by incubating for 30 min with his-resin, and washed 3 timeswith wash buffer (10 mM Imidazole in lysis buffer), and protein waseluted with elution buffer (250 mM Imidazole in lysis buffer).

Western Blot

Protein samples for Western immunoblot analysis were resolved bySDS-PAGE, transferred to nitrocellulose membrane, blocked inTris-buffered saline (TBS) containing 5% non-fat dry milk. Proteins werereacted with dog anti-E. canis or anti-E. chaffeensis serum (1:500).Blots were incubated with phosphatase-labeled goat anti-dog IgG dilutedin TBS (1:5000) (Kirkegaard & Perry, Gaithersburg, Mass.) and proteinsreactivity visualized after addition of alkaline phosphatase substrate(Kirkegaard & Perry).

ELISA

Sera from human patients infected with E. chaffeensis and dogs infectedwith E. canis were used to evaluate Ehrlichia chimera immunoreactivity.An ELISA was performed by incubating 50 ng/well of chimeric proteindiluted in PBS in 96-well ELISA plates (PolySorp, Nunc) and incubatedovernight at 4 C. The plates were washed 3× with 0.2% Tween 20 in TBS(TBST) and blocked 100 ul of blocking buffer (10% horse serum in TBST)and incubated at room temperature for 1 h with shaking. Plates werewashed 3× with TBST, and 50 ul of primary antibody diluted 1:100 inblocking buffer was incubated at room temperature for 1 h with shaking.Plates were washed 3× with TBST, add 50 ul of secondary antibodyAP-conjugate diluted in blocking buffer and incubate at room temperaturefor 1 h with shaking. Substrate was added and incubated at roomtemperature for 30 min with shaking in the dark and OD was determinedusing an ELISA plate reader @650 nm.

Results

Five Ehrlichia chimeric constructs were expressed in E. coli and therecombinant protein purified (FIGS. 1-5). Purified E. canis or E.chaffeensis specific chimeric proteins reacted with antibodies in doganti-E. canis or anti-E. chaffeensis (FIGS. 1-3). Similarly, E. canis/E.chaffeensis combination chimeric proteins reacted strongly with bothanti-E. canis and anti-E. chaffeensis dog sera demonstratingfunctionality of species specific epitopes represented in the chimera(FIG. 4 and FIG. 5). ELISA was performed with a panel of convalescentanti-E. chaffeensis patient sera to demonstrate immunoreactivity ofchimeric proteins with antibodies. The E. chaffeensis TRP32/TRP120/A34chimera reacted strongly with 10 HME patient sera demonstrating highimmunoreactivity with all HME patient sera (FIG. 1). Two E. canisspecifc chimeras (TRP140/TRP36/TRP19 and TRP36/TRP140) were reacted with10 sera from dogs naturally infected with E. canis. All sera from E.canis-infected dogs reacted strongly with the chimeric constructs (FIG.2 and FIG. 3). The E. chaffeensis/E. canis combination chimerasimmunoreactivity were tested with dog-anti-E. canis sera and humananti-E. chaffeensis patient sera. Both chimeras reacted strongly withanti-E. canis and anti-E. chaffeensis antibodies demonstrating thefunctionality of the species-specific epitopes represented within thechimera with E. canis and E. chaffeensis antibodies (FIG. 4 and FIG. 5).

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 9,545,439-   U.S. Pat. No. 9,545,439-   U.S. Pat. No. 10,117,921-   U.S. Pat. No. 10,117,921-   U.S. Pat. No. 10,117,921-   U.S. Pat. No. 10,238,736-   U.S. Pat. No. 4,220,450-   U.S. Pat. No. 4,373,932-   U.S. Pat. No. 4,472,509-   U.S. Pat. No. 4,897,268-   U.S. Pat. No. 4,938,948-   U.S. Pat. No. 5,075,109-   U.S. Pat. No. 5,440,013-   U.S. Pat. No. 5,446,128-   U.S. Pat. No. 5,470,723-   U.S. Pat. No. 5,470,932-   U.S. Pat. No. 5,543,504-   U.S. Pat. No. 5,552,157-   U.S. Pat. No. 5,565,213-   U.S. Pat. No. 5,567,434-   U.S. Pat. No. 5,618,914-   U.S. Pat. No. 5,656,016-   U.S. Pat. No. 5,670,155-   U.S. Pat. No. 5,697,899-   U.S. Pat. No. 5,738,868-   U.S. Pat. No. 5,741,516-   U.S. Pat. No. 5,770,219-   U.S. Pat. No. 5,779,708-   U.S. Pat. No. 5,783,208-   U.S. Pat. No. 5,795,587-   U.S. Pat. No. 5,797,898-   U.S. Pat. No. 5,840,833-   U.S. Pat. No. 5,853,744-   U.S. Pat. No. 5,859,184-   U.S. Pat. No. 5,891,506-   U.S. Pat. No. 5,929,237-   U.S. Pat. No. 6,136,610-   U.S. Pat. No. 6,210,708-   U.S. Pat. No. 6,372,445-   U.S. Pat. No. 6,617,142-   U.S. Pat. No. 6,875,750-   U.S. Pat. No. 6,951,765-   U.S. Pat. No. 7,163,677-   U.S. Pat. No. 7,282,194-   U.S. Pat. No. 7,344,893-   U.S. Pat. No. 7,371,582-   U.S. Pat. No. 8,580,280-   U.S. Pat. No. 8,980,288-   U.S. Pat. No. 8,980,288-   U.S. Pat. No. 9,662,385.-   U.S. Patent Appln. 2005/0047972-   U.S. Patent Appln. 2005/0065463-   U.S. Patent Appln. 2005/0250141-   U.S. Patent Appln. 2007/0264664-   U.S. Patent Appln. 2009/0005535-   U.S. Patent Appln. 2019/0008953-   U.S. Patent Appln. 2019/0038737-   EP2433646-   WO2005087803-   Aucouturier, et al., Expert Rev. Vaccines, 1, 111-118, 2002.-   Billeskov, et al., PLoS One 11, e0161217, 2016.-   Bonam et al., Trends in Pharmacological Sciences, 38(9): 771-778,    2017.-   Calabro et al., Vaccine, 31: 3363-3369, 2013.-   Carpino et al., Org. Proc. Res. Dev., 7(1)28-37, 2003.-   Didierlaurent, et al., J. Immunol., 183: 6186-6197, 2009.-   Didierlaurent, et al., J. Immunol., 193, 1920-1930, 2013-   Dumler et al., Clin. Infect. Dis., 45:S45-S51, 2007.-   Feng and Walker, Infect. Immun., 72:966-971, 2004.-   Fishbein et al., Human ehrlichiosis in the United States, 1985    to 1990. AnnInternMed 120:736-743, 1994.-   Garcon and Van Mechelen, Expert Rev. Vaccines, 10, 471-486, 2011.-   Geysen et al., Proc. Natl. Acad. Sci. USA, 81(13):3998-4002, 1984.-   He et al., Vaxign: the first web-based vaccine design program for    reverse vaccinology and applications for vaccine development. J    Biomed Biotechnol 2010:297505, 2010.-   Koczula et al., 2016.-   Künzi, et al., Vaccine, 27, 3561-3567, 2009.-   Kuriakose et al., Ehrlichia chaffeensis transcriptome in mammalian    and arthropod hosts reveals differential gene expression and post    transcriptional regulation. PLoS One 6:e24136, 2011.-   Kuriakose et al., Molecular basis of antibody mediated immunity    against Ehrlichia chaffeensis involves species-specific linear    epitopes in tandem repeat proteins. Microbes Infect 14:1054-1063,    2012.-   Li and Winslow, Survival, replication, and antibody susceptibility    of Ehrlichia chaffeensis outside of host cells. InfectImmun    71:4229-4237, 2003.-   Li et al., Antibodies highly effective in SCID mice during infection    by the intracellular bacterium Ehrlichia chaffeensis are of    picomolar affinity and exhibit preferential epitope and isotype    utilization. J Immunol 169:1419-1425, 2002.-   Li et al., Outer membrane protein-specific monoclonal antibodies    protect SCID mice from fatal infection by the obligate intracellular    bacterial pathogen Ehrlichia chaffeensis. J Immunol 166:1855-1862,    2001.-   Lin et al., Global proteomic analysis of two tick-borne emerging    zoonotic agents: Anaplasma phagocytophilum and Ehrlichia    chaffeensis. Front Microbiol 2:24, 2011.-   Magnan et al., High-throughput prediction of protein antigenicity    using protein microarray data. Bioinformatics 26:2936-2943, 2010.-   McBride and Walker, Progress and obstacles in vaccine development    for the ehrlichioses. Expert Rev Vaccines 9:1071-1082, 2010.-   Mizuno et al., Chemistry. 23(58):14394-14409, Oct. 17 2017.-   Morel, S. et al., Vaccine, 29, 2461-2473, 2011.-   Nandi et al., CD4 T-cell epitopes associated with protective    immunity induced following vaccination of mice with an ehrlichial    variable outer membrane protein. InfectImmun 75:5453-5459, 2007.-   Olano et al., Human monocytotropic ehrlichiosis, Missouri.    EmergInfectDis 9:1579-1586, 2003.-   Paparone et al., Ehrlichiosis with pancytopenia and ARDS. New Jersey    Med 92:381-385, 1995.-   Paterson et al., Anal Chem. 86(19):9481-8, October 7; 2014.-   Pierce Immunotechnology Catalog and Handbook, at A12-A13, 1991-   Racine et al., IgM production by bone marrow plasmablasts    contributes to long-term protection against intracellular bacterial    infection. J Immunol 186:1011-1021, 2011.-   Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,    1289-1329, 1990.-   Sotomay et al., Animal model of fatal human monocytotropic    ehrlichiosis. AmJPath 158:757-769, 2001.-   Stuen et al., Acta Vet Scand., 57:40, 2015.-   Tandrup Schmidt, et al., Pharmaceutics, 8, 7, 2016.-   The Science and Practice of Pharmacy, 21^(st) Ed. Lippincott    Williams and Wilkins, 2005.-   Tollersrud et al., Vaccine, 19:3896-3903, 2001.-   Vega et al., Vaccine, 25:519-525, 2007.-   Walker and Dumler, Human monocytic and granulocytic ehrlichioses.    Discovery and diagnosis of emerging tick-borne infections and the    critical role of the pathologist. [Review] [50 refs]. Archives of    Pathology & Laboratory Medicine 121:785-791, 1997.-   Walker et al., Ehrlichia chaffeensis: a prevalent, life-threatening,    emerging pathogen. Trans Am Clin Climatol Assoc 115:375-382;    discussion 382-374, 2004.-   Wang et al., 2017.-   Winslow et al., Ann. NY Acad. Sci., 990:435-443, 2003.-   Winslow et al., Infect. Immun., 68:2187-2195, 2000.-   Winslow et al., Infection of the laboratory mouse with the    intracellular pathogen Ehrlichia chaffeensis. InfectImmun    66:3892-3899, 1998.-   Yager et al., Infect. Immun., 73:8009-8016, 2005.-   Zemella et al., Cell-Free Protein Synthesis: Pros and Cons of    Prokaryotic and Eukaryotic Systems. Chembiochem.; 16(17):2420-2431,    2015.

1. An isolated polypeptide, wherein the isolated polypeptide comprises:(i) at least two of the immunogenic sequences of Table 1, or a sequenceat least 90% identical; and (ii) wherein at least one of the immunogenicsequences is contiguously repeated in the polypeptide.
 2. The isolatedpolypeptide of claim 1, wherein each of the at least two immunogenicsequences of Table 1, or a sequence at least 90% identical arecontiguously repeated 1, 2, 3, 4, 5, 6, or 7 times in the polypeptide.3. The isolated polypeptide of claim 1, wherein the isolated polypeptidecomprises one or more of (SEQ ID NOs:11-16 or 36-42), wherein the one ormore of (SEQ ID NOs:11-16 or 36-42) are contiguously repeated 0, 1, 2,or 3 times.
 4. The isolated polypeptide of claim 1, wherein each of theimmunogenic sequences are contiguously repeated from 1 to 3 times in thepolypeptide.
 5. The isolated polypeptide of claim 4, wherein each of theimmunogenic sequences are contiguously repeated from 1 to 2 times in thepolypeptide.
 6. The isolated peptide of claim 1, wherein the isolatedpolypeptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all ofthe following immunogenic sequences: TRP120 (SEQ ID NO:22), TRP140 (SEQID NO:23), A34N1 (SEQ ID NO:7), TRP63 (SEQ ID NO:18), TRP47 (SEQ IDNO:17), TRP75 (SEQ ID NO:19), TRP28 (SEQ ID NO:2), TRP36R1 (SEQ IDNO:3), TRP36R2 (SEQ ID NO:4), TRP36R3 (SEQ ID NO:6), TRP36CO (SEQ IDNO:36), TRP19 (SEQ ID NO:1), HSP (SEQ ID NO:24), or a sequence at least90% identical; wherein each of the immunogenic sequences arecontiguously repeated from 1 to 7 times in the polypeptide.
 7. Thepolypeptide of claim 1, wherein the isolated polypeptide comprisesTRP36R1 and TRP140.
 8. The polypeptide of claim 7, wherein the TRP36R1is contiguously repeated 4-8 times, and wherein the TRP140 iscontiguously repeated 1-3 times.
 9. The polypeptide of claim 8, whereinthe polypeptide comprises or consists of 8 repeats of TRP36R1 and 4repeats of TRP140.
 10. The polypeptide of claim 9, wherein thepolypeptide comprises or consists of SEQ ID NO:27.
 11. The polypeptideof claim 7, wherein the polypeptide further comprises TRP19.
 12. Thepolypeptide of claim 11, wherein the polypeptide comprises or consistsof SEQ ID NO:26.
 13. The polypeptide of claim 1, wherein the isolatedpolypeptide comprises at least two, at least three, at least four, atleast five or all of the immunogenic sequences: TRP32, TRP120, TRP36(such as TRP36R1, TRP36R2, TRP36R3, or TRP36CO), TRP140, TRP28, and/orHSP.
 14. The polypeptide of claim 13, wherein the TRP36R1 if present isrepeated 2-6 times, and wherein the other immunogenic sequences arerepeated 1-3 times.
 15. The polypeptide of claim 1, wherein thepolypeptide comprises all of TRP32, TRP120, TRP36, TRP140, TRP28, andHSP.
 16. The polypeptide of claim 15, wherein the polypeptide comprisesor consists of SEQ ID NO:28.
 17. The polypeptide of claim 13, whereinthe polypeptide comprises TRP120, TRP36, TRP140, and TRP28.
 18. Thepolypeptide of claim 17, wherein the polypeptide comprises or consistsof SEQ ID NO:29.
 19. The polypeptide of claim 1, wherein the isolatedpolypeptide comprises at least three, at least four, at least five orall of TRP32R1, TRP32R2, TRP32R3, TRP32R4, TRP120, and A34N1.
 20. Thepolypeptide of claim 19, wherein TRP120 and A34N1 are each contiguouslyrepeated 1-3 times.
 21. The polypeptide of claim 20, wherein TRP120 andA34N1 are each contiguously repeated 2 times.
 22. The polypeptide ofclaim 21, wherein the polypeptide comprises or consists of SEQ ID NO:25.23-49. (canceled)
 50. The polypeptide of claim 1, wherein thepolypeptide comprises a polypeptide of any one of SEQ ID NOs: 25-35.51-58. (canceled)
 59. The polypeptide of claim 1, wherein the differentimmunogenic sequences are not separated by a linker or a spacer.
 60. Thepolypeptide of claim 1, wherein the different immunogenic sequences areseparated by a linker or a spacer.
 61. The polypeptide of claim 60,wherein the linker is a glycine linker.
 62. The polypeptide of claim 61,wherein the glycine linker has the amino acid sequence -(G)x-, whereinX=3-5.
 63. The polypeptide of claim 1, wherein the polypeptide is lessthan 500, less than 450, less than 400, less than 350, less than 300,less than 250, less than 200, or less than 150 amino acids in length.64. The polypeptide of claim 1, wherein the polypeptide is comprised ina pharmaceutical preparation.
 65. The pharmaceutical preparation ofclaim 64, wherein the pharmaceutical preparation is formulated forparenteral, intravenous, subcutaneous, intranasal, sublingual, orintradermal administration.
 66. The polypeptide of claim 1, wherein thepolypeptide is attached to a solid support or is comprised in adiagnostic kit.
 67. The polypeptide of claim 66, wherein the solidsupport is glass or plastic.
 68. The polypeptide of claim 66, whereinthe solid support is comprised in a lateral flow assay, or microfluidicdevice.
 69. An isolated polypeptide of Formula I(A_(s)-B_(t)-C_(u)-D_(v)-E_(w)-F_(x)-G_(y)-H_(z))_(n), wherein each ofA, B, C, D, E, F, G, and H is a peptide selected from SEQ ID NOs:1-24and 36-42, or a sequence at least 90% identical to any one of (SEQ IDNOs:1-24 or 36-42), wherein s, t, u, v, x, y, and z is an integer 0-8,wherein at least two of s-z are ≥1 and at least one of s-z is ≥2, andwherein n is an integer 1-5. 70-79. (canceled)
 80. A pharmaceuticalpreparation comprising the polypeptide of claim 1 and a pharmaceuticallyacceptable excipient. 81-112. (canceled)
 113. A nucleic acid encodingthe polypeptide of claim
 1. 114-115. (canceled)
 116. A host cellcomprising the nucleic acid of claim
 113. 117. (canceled)
 118. A methodof detecting antibodies that specifically bind an Ehrlichia organism ina test sample, comprising: (a) contacting an isolated polypeptide ofclaim 1; (b) detecting the peptide-antibody complexes; wherein thedetection of the peptide-antibody complexes is an indication thatantibodies specific for an Ehrlichia organism are present in the testsample, and wherein the absence of the peptide-antibody complexes is anindication that antibodies specific an Ehrlichia organism are notpresent in the test sample. 119-122. (canceled)
 123. A method ofidentifying an Ehrlichia infection in a mammalian subject comprising:(a) contacting a biological sample from the subject with an isolatedpolypeptide of claim 1 under conditions that allow peptide-antibodycomplexes to form; and (b) detecting the peptide-antibody complexes;wherein the detection of the peptide-antibody complexes is an indicationthat the subject has an Ehrlichia infection. 124-126. (canceled)
 127. Akit comprising: (a) the isolated polypeptide of claim 1, (b) an anti-dogor anti-human secondary antibody linked to a reporter molecule; and, (c)an appropriate reagent for detection of the reporter molecule. 128-133.(canceled)
 134. A method of inducing an immune response in a mammaliansubject comprising administering to the subject an effective amount of apharmaceutical preparation comprising the polypeptide of claim 1.135-136. (canceled)
 137. A method of treating an Ehrlichia chaffeensisor Ehrlichia canis infection in a subject comprising: (a) contacting abiological sample from the subject with an isolated polypeptide of claim1 under conditions that allow peptide-antibody complexes to form; (b)detecting the peptide-antibody complexes; wherein the detection of thepeptide-antibody complexes is an indication that the subject has anEhrlichia chaffeensis or Ehrlichia canis infection; and (c)administering a therapeutic compound to treat Ehrlichia infection in thesubject. 138-142. (canceled)